Antithrombotic amidinotetrahydropyridylalanine derivatives

ABSTRACT

This invention is directed to compounds of formula (I), ##STR1## pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates of either entity, wherein Y is optionally monounsaturated C 3  -C 5  alkylene optionally substituted with C 1  -C 4  alkyl or methylene; R 1  is H; C 1  -C 4  alkyl optionally substituted with C 1  -C 4  alkoxy, OH, NR 5  R 6 , CONR 5  R 6 , C 3  -C 6  cycloalkyl or aryl; or C 3  -C 6  alkenyl; R 2  is H; C 1  -C 4  alkyl optionally substituted with C 1  -C 4  alkoxy, OH, NR 5  R 6 , CONR 5  R 6 , C 3  -C 6  cycloalkyl or aryl; or CONR 5  R 6  ; R 3  and R 4  are each independently selected from H; C 1  -C 4  alkyl optionally substituted with NR 5  R 6  ; C 1  -C 4  alkoxy; halo; CONR 5  R 6  and aryl; aryl is phenyl optionally substituted with one, two or three substituents independently selected from C 1  -C 4  alkyl, C 1  -C 4  alkoxy, OH, halo and CF 3  ; R 5  and R 6  are each independently selected from H and C 1  -C 4  alkyl; and m and n are each independently 1, 2 or 3; which are potent and selective thrombin inhibitors useful in the treatment of inter alia, deep vein thrombosis; reocclusion following thrombolytic therapy; chronic arterial obstruction; peripheral vascular disease; acute myocardial infarction; unstable angina; atrial fibrillation; thrombotic stroke; transient ischaemic attacks; restenosis and occlusion following angioplasty; or neurodegenerative disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This invention is the national stage of international patent applicationnumber PCT/EP96/01459, filed Apr. 1, 1996, entitled "AntithromboticAmidinotetrahydropyridylalanine Derivatives".

This invention relates to a series of amidinotetrahydropyridylalaninederivatives, which are antithrombotic agents, having utility in avariety of therapeutic areas including the prevention and/or treatmentof deep vein thrombosis (DVT) after surgery, major medical illness,paralysis, malignancy, prolonged immobilisation trauma, application oflower limb plaster casts, or fractures of the lower limbs or pelvis;recurrent DVT; DVT during pregnancy when there is a previous historythereof; reocclusion following thrombolytic therapy; chronic arterialobstruction; peripheral vascular disease; acute myocardial infarction;unstable angina; atrial fibrillation; thrombotic stroke; transientischaemic attacks; disseminated intravascular coagulation; coagulationin extra-corporeal circuits; occlusion of arterio-venous shunts andblood vessel grafts (including coronary artery by-pass grafts); andrestenosis and occlusion following angioplasty. They also have utilityas an adjunct to thrombolytic therapy.

The compounds of the invention are potent and selective inhibitors ofthrombin, which is the final serine protease enzyme in the coagulationcascade. The prime function of thrombin is the cleavage of fibrinogen toproduce fibrin which forms linear insoluble polymers which, in turn, arecross-linked by factor XIIIa, itself activated by thrombin. In addition,thrombin regulates its own production by activation of factors V andVIII earlier in the cascade. It also has important actions at thecellular level, where it acts on specific receptors to cause plateletaggregation, endothelial cell activation and fibroblast proliferation.Thus thrombin has a central regulatory role in homeostasis and thrombusformation.

Clearly then, potent, selective and orally bioavailable thrombininhibitors represent an attractive target for the convenient therapeuticcontrol of thrombosis. In addition, thrombin potently causes neuriteretraction and therefore a thrombin inhibitor is of potentialtherapeutic utility in the treatment of acute and chronicneurodegenerative disorders. Furthermore, the compounds disclosed hereinare of potential value in the treatment of inflammatory disorders andscarring, and in wound healing.

Because of their potential as substrate mimics, arginine derivativeshave been explored as thrombin inhibitors and this work led to thediscovery of argatroban (see Cardiovascular Drug Rev., 1991, 9, 247). Inturn, other research groups have sought to express the basic argininefunction in a variety of alternative structures; for example,WO-A-92/08709 discloses amidino, guanidino, amidoximino, aminomethyl andamino phenylalanine derivatives as antithrombotic agents.

The compounds of the present invention are significantly more potentthrombin inhibitors than those mentioned above, selective (in comparisonwith their inhibition of, for example, trypsin, plasmin,butyrylcholinesterase and elastase), well tolerated and orallybioavailable.

Accordingly, the present invention provides a compound of formula (I):##STR2## pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable solvates of either entity,

wherein

Y is optionally monounsaturated C₃ -C₅ alkylene optionally substitutedwith C₁ -C₄ alkyl or methylene;

R¹ is H; C₁ -C₄ alkyl optionally substituted with C₁ -C₄ alkoxy, OH, NR⁵R⁶, CONR⁵ R⁶, C₃ -C₆ cycloalkyl or aryl; or C₃ -C₆ alkenyl;

R² is H; C₁ -C₄ alkyl optionally substituted with C₁ -C₄ alkoxy, OH, NR⁵R⁶, CONR⁵ R⁶, C₃ -C₆ cycloalkyl or aryl; or CONR⁵ R⁶ ;

R³ and R⁴ are each independently selected from H; C₁ -C₄ alkyloptionally substituted with NR⁵ R⁶ ; C₁ -C₄ alkoxy; halo; CONR⁵ R⁶ andaryl;

R⁵ and R⁶ are each independently selected from H and C₁ -C₄ alkyl; and

m and n are each independently 1, 2 or 3.

In the above definition, aryl means phenyl optionally substituted withone, two or three substituents independently selected from C₁ -C₄ alkyl,C₁ -C₄ alkoxy, OH, halo and CF₃ ; halo means fluoro, chloro, bromo oriodo. Unless otherwise indicated, alkyl and alkoxy groups having threeor more carbon atoms and alkenyl groups having four or more carbon atomsmay be straight-chain or branched-chain.

The compounds of formula (I) contain two or more asymmetric centres andthus can exist as stereoisomers, i.e. as enantiomers or asdiastereoisomers, and the invention includes both the separatedindividual stereoisomers as well as mixtures thereof.

The preferred stereoisomers are of formula (IA): ##STR3##

Also included in the invention are radiolabelled derivatives ofcompounds of formula (I) which are suitable for biological studies.

The pharmaceutically acceptable salts of the compounds of formula (I)are, for example, non-toxic acid addition salts formed with inorganicacids such as hydrochloric, hydrobromic, sulphuric and phosphoric acid,with organo-carboxylic acids, or with organo-sulphonic acids. Compoundsof formula (I) can also provide pharmaceutically acceptable metal salts,in particular non-toxic alkali metal salts, with bases. Examples includethe sodium and potassium salts.

A preferred group of compounds of formula (I) is that wherein Y isoptionally monounsaturated C₄ alkylene substituted with methyl or ethyl;R¹ is C₁ -C₄ alkyl; R² is H; R³ and R⁴ are H; and m and n are eachindependently 1 or 2.

A more preferred group of compounds is that of formula (IB): ##STR4##wherein--represents an optional carbon-carbon single bond; R¹ and R⁷ aremethyl; m is 1 or 2; and n is 2.

Particularly preferred individual compounds of the invention include:

4-methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid;

4-methyl-1-N-(3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid; and

4(R)-methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid;

and pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable solvates of either entity.

In another aspect, the present invention provides processes for thepreparation of compounds of formula (I) and their pharmaceuticallyacceptable salts.

A compound of formula (I) may be prepared by hydrolysis of its loweralkyl ester precursor of formula (II): ##STR5## wherein R⁸ is C₁ -C₃alkyl, preferably methyl or ethyl, and Y, R¹, R², R³, R⁴, m and n are aspreviously defined for formula (I). The reaction may be acid- orbase-catalysed, but is generally carried out using an alkali metalhydroxide such as sodium or potassium hydroxide in aqueous solution,optionally in the presence of a suitable cosolvent, at from about 0° C.to about 100° C. Preferred conditions are the use of aqueous sodiumhydroxide solution at from about 0° C. to about room temperature.

The novel intermediate esters of formula (II) also form part of theinvention.

A compound of formula (II) may be prepared from a compound of formula(III): ##STR6## wherein P is a conventional amine protecting group andY, R¹, R², R³, R⁴, R⁸, m and n are as previously defined for formula(II). This may be achieved by double-deprotection of the protectedamidine group under standard conditions. When P is the preferredprotecting group, i.e. t-butoxycarbonyl (Eoc), deprotection may beeffected under acidic conditions, e.g. using trifluoroacetic acid orhydrogen chloride in a suitable solvent. Preferably the reaction isconducted using hydrogen chloride in dichloromethane at from about 0° C.to about room temperature.

A compound of formula (III) may be prepared by N-alkylation of acompound of formula (IV): ##STR7## wherein P, Y, R², R³, R⁴, R⁸, m and nare as previously defined for formula (III), with the proviso that noneof the other nucleophilic centres within (IV) provides a more reactivealkylation site, e.g. in certain cases where R², R³ or R⁴ is C₁ -C₄alkyl substituted with NR⁵ R⁶.

In general, the alkylation may be achieved by reaction of a compound offormula (IV) with a compound of formula R¹ Q, wherein R¹ is aspreviously defined for formula (III) and Q is a suitable leaving group,e.g. halo, C₁ -C₄ alkanesulphonyloxy, trifluoromethanesulphonyloxy orarylsulphonyloxy (such as benzenesulphonyloxy or p-toluenesulphonyloxy),in the presence of an appropriate base, e.g. the carbonate orbicarbonate salt of an alkali or alkaline earth metal, in a suitablesolvent such as a C₁ -C₃ alkanol, acetonitrile, dimethylformamide orN,N-dimethylacetamide, optionally in the presence of the iodide salt ofsodium or potassium, at from about room temperature to about 100° C.Preferably Q is chloro, bromo or iodo, the base is sodium or potassiumcarbonate or bicarbonate, the solvent is acetonitrile and the reactionis conducted at about 80°-85° C.

When R¹ is methyl, the N-methylation can be conveniently carried out bya reductive alkylation procedure wherein (IV) is treated with aqueousformaldehyde solution followed by an appropriate reducing agent in asuitable solvent. Preferably both reaction steps are conducted at roomtemperature in dichloromethane as solvent, with sodiumtriacetoxyborohydride being employed in the reduction step.

A compound of formula (IV) may be prepared by N-deprotection of acompound of formula (V): ##STR8## wherein P¹ is a protecting group andP, Y, R², R³, R⁴, R⁸, m and n are as previously defined for formula(IV). P¹, which represents a conventional amine protecting group, ischosen with due regard to its compatibility with the various reagentsemployed in earlier synthetic steps of the over-all process and also tothe reaction conditions required for its selective removal; preferably,it is trifluoroacetyl. The particular protecting group can be removedunder standard conditions which, in the case of trifluoroacetyl, aremild aqueous base optionally in the presence of a C₁ -C₃ alkanol ascosolvent. Preferred conditions are sodium or potassium carbonate inaqueous methanol or ethanol at about room temperature.

A compound of formula (V) may be prepared from a compound of formula(VI): ##STR9## wherein P¹, Y, R², R³, R⁴, R⁸, m and n are as previouslydefined for formula (V), by reaction with a S-alkylisothioureaderivative of formula (VII): ##STR10## wherein R⁹ is C₁ -C₃ alkyl and Pis as previously defined for formula (V), in a suitable solvent,optionally in the presence of a mercury(II) salt, at from about 0° C. toabout room temperature.

A compound of formula (VI) may be prepared from a compound of formula(VIII): ##STR11## wherein P¹, Y, R², R³, R⁴, R⁸, m and n are aspreviously defined for formula (VI), using a standard N-dealkylationreagent. In a preferred procedure, the debenzylation is effected by, ina first step, reaction of (VIII) with 1-chloroethyl chloroformate,optionally in the presence of 1,8-bis(dimethylamino)naphthalene, in asuitable solvent such as dichloromethane, 1,2-dichloroethane or toluene,at from about 0° C. to about 110° C., followed by a second step in whichthe intermediate carbamate is heated under reflux with a C₁ -C₃ alkanol.Preferably, in the first step the solvent is dichloromethane and thereaction temperature is at from about 0° C. to about room temperature,while in the second step the alkanol is methanol or ethanol.

A compound of formula (VIII) may be assembled by a number of strategies.One approach may involve the coupling of a compound of formula (IX):##STR12## wherein P¹, R², R³, R⁴, m and n are as previously defined forformula (VIII), with a compound of formula (X): ##STR13## wherein Y andR⁸ are as previously defined for formula (VIII). The coupling reactionmay be achieved using conventional amide bond-forming techniques. Forexample, the acid may be activated by formation of the correspondingacyl halide, e.g. bromide or chloride, followed by reaction of thelatter with an amine of formula (X), optionally in the presence of areaction-inert base to act as acid scavenger, in a suitable solvent suchas dichloromethane. Alternatively, any of a host of peptide couplingvariations may be used. For example, the acid may be activated using acarbodiimide such as 1,3-dicyclohexylcarbodiimide or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, optionally in thepresence of 1-hydroxybenzotriazole and, where appropriate, areaction-inert amine such as N-methylmorpholine orN-ethyidiisopropylamine (for example when either (X) or the carbodiimideis in the form of an acid addition salt) and/or a catalyst such as4-dimethylaminopyridine, in a suitable solvent such as dichloromethane.Thus, in a preferred process, (IX) is converted to the correspondingacyl chloride using oxalyl chloride and a catalytic amount ofdimethylformamide in a suitable solvent, e.g. dichloromethane, at about0°-5° C. and then the acyl chloride is reacted with (X), optionally as asuitable acid addition salt, in the presence of N-ethyldiisopropylamineas reaction-inert base in dichloromethane at from about 0° C. to aboutroom temperature.

A compound of formula (IX) may be prepared from a corresponding ester offormula (XI): ##STR14## wherein R¹⁰ is C₁ -C₄ alkyl or benzyl, and P¹,R², R³, R⁴, m and n are as previously defined for formula (IX).Preferably R¹⁰ is t-butyl.

The conversion of (XI) to (IX) can be effected by standard hydrolytic orO-dealkylation procedures and will be dependent on the nature of P¹. Forexample, when R¹⁰ is t-butyl and P¹ is trifluoroacetyl, protonolysisusing a reagent such as trifluoroacetic acid or hydrogen chloride in asuitable solvent is the method of choice. Preferably the reaction isconducted using hydrogen chloride in dichloromethane at from about 0° C.to about room temperature.

A compound of formula (XI) may be prepared by sulphonylation of acompound of formula (XII): ##STR15## wherein R¹⁰ is as previouslydefined for formula (XI), optionally as a suitable acid addition salt,with a compound of formula (XIII): ##STR16## wherein Z is halo,preferably chloro, and P¹, R², R³, R⁴, m and n are as previously definedfor formula (XI), under conventional conditions in the presence of areaction-inert base in a suitable solvent at from about 0° C. to aboutroom temperature. Preferably the base is N-ethyldiisopropylamine and thesolvent is dichloromethane.

A compound of formula (XII) may be prepared by N-deprotection of acompound of formula (XIV): ##STR17## wherein R¹⁰ is as previouslydefined for formula (XII) and P is as previously defined for formula(VII). As for a compound of formula (III), P is preferably Boc and (XIV)may be similarly deprotected. However, when R¹⁰ is the preferred group(t-butyl), the deprotection method of choice involves the use of neatformic acid at about room temperature.

A compound of formula (XIV) may be prepared by partial reduction of acompound of formula (XV): ##STR18## wherein P and R¹⁰ are as previouslydefined for formula (XIV) and Z is as previously defined for formula(XIII) but is preferably bromo, using a conventional reducing agent in aC₁ -C₃ alkanol. A preferred method employs sodium borohydride in ethanolat about 0° C.

A compound of formula (XV) may be prepared by N-benzylation of acompound of formula (XVI): ##STR19## wherein P and R¹⁰ are as previouslydefined for formula (XV), using the appropriate benzyl halide in asuitable solvent under standard conditions, e.g. benzyl bromide inethanol at about room temperature.

The N-protected α-amino ester of formula (XVI) is obtainable from thecorresponding α-amino acid, 3-(3-pyridyl)alanine (see Int. J. Pept.Prot. Res., 1987, 29, 118 for S- and R-enantiomers), using conventionalamino acid protecting group chemistry. This may be achieved, forexample, by effecting N-protection initially, followed by esterificationof the carboxyl group using the appropriate alcohol of formula R¹⁰ OH.

A compound of formula (XIII) may be prepared from a compound of formula(XVII): ##STR20## wherein P¹, R², R³, R⁴, m and n are as previouslydefined for formula (XIII), by the application of known methods for theelectrophilic introduction of a SO₂ Z group, wherein Z is as previouslydefined for formula (XIII), into an aromatic ring system. For example,when Z is chloro, by the action of chlorosulphonic acid at from about-15° C. to about room temperature.

A compound of formula (XVII) may be prepared from a compound of formula(XVIII): ##STR21## wherein R², R³, R⁴, m and n are as previously definedfor formula (XVII), by conventional procedures. For example, when P¹ istrifluoroacetyl, by using trifluoroacetic anhydride, optionally in thepresence of a base such as N-methylmorpholine or N-ethyldiisopropylamineand a solvent such as dichloromethane at from about 0° C. to about roomtemperature.

A compound of formula (X) may be prepared by a variety of methods, e.g.by standard cyclic α-amino acid/ester syntheses, and, when a particularstereoisomer is required, by classical resolution procedures or byasymmetric synthesis.

For example, when (X) represents a compound of formula (XA): ##STR22##wherein R⁷ is as previously defined for formula (IB) and R⁸ is aspreviously defined for formula (X), 4-methylpyridine offers a convenientstarting point and may be processed as follows. Followingquarternisation by alkylation, e.g. using a conventional methylationprocedure, the resulting pyridinium salt is subjected to partialreduction using sodium borohydride followed by in situ α-cyanation ofthe 2,5-dihydropyridine intermediate using hydrogen cyanide to afford,in this example,1-methyl-2(R,S)-cyano-4-methyl-1,2,3,6-tetrahydropyridine. Next, thenitrile is converted to the required ester derivative andN-demethylation effected using a suitable chloroformate, e.g.2,2,2-trichloroethyl chloroformate. Finally, N-deprotection is carriedout using the appropriate reagent, e.g. zinc dust in this example.

An alternative approach to a compound of formula (XA) involves azaDiels-Alder cycloaddition chemistry in which an imine of formula (XIX),wherein P² is a suitable protecting group, e.g. benzyl, is reacted witha diene of formula (XX): ##STR23## (see Tetrahedron:Asymmetry,1991,2,1263), followed by N-debenzylation, again using a chloroformatereagent, e.g. 1-chloroethyl chloroformate in this case. The finalN-deprotection may be achieved using excess alcohol (R⁸ OH) at aboutreflux temperature.

Clearly, reduction of (XA), e.g. by catalytic hydrogenation, willprovide the corresponding piperidine-2-carboxylic esters.

When P² also acts as a chiral auxiliary, e.g. it is 1(R)- or1(S)-phenylethyl, a useful degree of asymmetric induction is achievablein the (4+2) cycloaddition reaction affording a mixture of easilyseparable diastereoisomers; the 1(S)-auxiliary induces(R)-stereochemistry at the 2-position and the 1(R)-auxiliary providesthe antipodal series (see Tetrahedron:Asymmetry, 1991, 2,1263 andTetrahedron, 1992,48,9707). N-Deprotection may be effected as above forthe case wherein P² is benzyl, thus providing either the preferred 2(R)-or the 2(S)-enantiomer of (XA) respectively.

Again, catalytic hydrogenation of these enantiomers should lead to the(2R,4S)- and (2S,4R)-piperidine enantiomers respectively.

Alternative approaches to these piperidine enantiomers wherein R⁷ ismethyl, and also to the corresponding (2R,4R)- and (2S,4S)-enantiomers,are described in Biochem.Biophys.Res.Comm., 1981,101,440, in whichclassical fractional distillation, fractional crystallisation of saltsformed from optically active acids (L- and D-tartaric acid) andepimerisation techniques are employed.

Resolution may also be achieved by chromatographic separationprocedures. For example, acid-catalysed hydrolysis of the cycloadductformed from (XIX) wherein P² is benzyl, and (XX), affords1-benzyl-4-methyl-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylic acidwhich is then esterified under standard conditions with a chiralalcohol, e.g. a N-protected ephedrine derivative such asN-acetyl-(1R,2S)-ephedrine. N-Deprotection using, for example,2,2,2-trichloroethyl chloroformate followed by zinc dust as describedabove, followed by chromatography on silica gel, furnishes theindividual 2(R)- and 2(S)-diastereoisomeric esters, each of which isprocessed as followed. N-Reprotection, e.g. using a Boc group, removalof the chiral auxiliary by base-catalysed hydrolysis, reesterificationwith R⁸ OH, and removal of the Boc group, provides the 2(R)- and2(S)-enantiomers of (XA) whose identities can be confirmed by comparisonwith the enantiomers obtained by the asymmetric aza Diels-Alderchemistry previously described.

An alternative approach for assembling a compound of formula (VIII) mayinvolve the sulphonylation of a compound of formula (XXI): ##STR24##wherein Y and R⁸ are as previously defined for formula (VIII), with acompound of formula (XIII), under the conditions employed for reacting(XIII) with (XII).

A compound of formula (XXI) may be prepared from a compound of formula(XXII): ##STR25## wherein P is as previously defined for formula (XVI)and Y and R⁸ are as previously defined for formula (XXI), by analogywith the reactions involved in the conversion of (XVI) to (XIV),followed by N-deprotection as already described for the conversion of(III) to (II).

A compound of formula (XXII) may be prepared by coupling theappropriately N-protected 3-(3-pyridyl)alanine with a compound offormula (X), using the conditions already described for coupling (X)with (IX). In a preferred process, the reagents employed are1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazoleand N-methylmorpholine, in dichloromethane as solvent, at from about 0°C. to about room temperature.

The bicyclic amines of formula (XVIII) and intermediates employed in thepreparation thereof, when neither commercially available norsubsequently described, can be obtained either by analogy with theprocesses described in the Preparations section or by conventionalsynthetic procedures, in accordance with standard textbooks on organicchemistry or literature precedent, from readily accessible startingmaterials using appropriate reagents and reaction conditions.

Moreover, persons skilled in the art will be aware of variations of, andalternatives to, those processes described hereinafter in the Examplesand Preparations sections which allow the compounds defined by formula(I) to be obtained.

The pharmaceutically acceptable acid addition salts of the compounds offormula (I) may also be prepared in a conventional manner. For example asolution of the free base is treated with the appropriate acid, eitherneat or in a suitable solvent, and the resulting salt isolated either byfiltration or by evaporation under vacuum of the reaction solvent.Pharmaceutically acceptable base addition salts can be obtained in ananalogous manner by treating a solution of a compound of formula (I)with the appropriate base. Both types of salt may be formed orinterconverted using ion-exchange resin techniques.

The biological activities of the compounds of the present invention weredetermined by the following test methods.

Chromogenic Assays

The inhibition of thrombin, trypsin, plasmin or factor Xa is measured in96 well plate chromogenic assays. The percentage inhibition and IC₅₀ arecalculated from triplicate samples of an 8 concentration dose-responsecurve. From the substrate Km and IC₅₀, the Ki for each inhibitor iscalculated. All assays are carried out in a total incubation of 200 μlof 50 mM HEPES and 150 mM NaCl at pH 8.0, and all compound dilutions arepreincubated with enzyme at room temperature for 15 minutes prior toaddition of substrate. After 30 minutes incubation at 30° C., the O.D.is measured at 405 nM in a 96 well plate reader. Thrombin activity ismeasured using bovine thrombin and S2238 (H-D-Phe-Pip-Arg-pNA), bovinepancreatic trypsin is assayed with S2222 (Benz-Isoleu-Glu-Gly-Arg-pNA),bovine plasma plasmin is assayed with Chromozym PL(Tosyl-Gly-Pro-Lys-pNA) and bovine fac or Xa is assayed in 50 mM Tris,150 mM NaCl, pH 7.5 buffer with S2222.

Clotting Assays

Thrombin time (TT) and activated partial thromboplastin time (APTT) aremeasured using Instrumentation Laboratories (IL) Test TT reagent and ILTest APTT (ellagic acid) reagent respectively in an AutomatedCoagulation Laboratory (ACL), according to the manufacturer'sinstructions.

In Vitro

To 1 ml aliquots of rat pooled plasma (citrated), a 1/100 volume of arange of compound concentrations is added and preincubated at roomtemperature for 15 minutes, after which the TT and APTT are measured.

Ex Vivo

Compounds are dosed per os, intravenously or intraduodenally to rats.Pre- and post-dose blood samples are taken into citrate solution andplasma prepared. TT and APTT are measured as for in vitro assays.

In therapy, the compounds of formula (I), their pharmaceuticallyacceptable salts, and pharmaceutically acceptable solvates of eitherentity, can be administered alone, but will generally be administered inadmixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.Preferably, they are administered orally in the form of tabletscontaining such excipients as starch or lactose, or in capsules orovules either alone or in admixture with excipients, or in the form ofelixirs, solutions or suspensions containing flavouring or colouringagents. They can also be injected parenterally, for exampleintravenously, intramuscularly or subcutaneously. For parenteraladministration, they are best used in the form of a sterile aqueoussolution which may contain other substances, for example enough salts orglucose to make the solution isotonic with blood. For buccal orsublingual administration they may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

For oral, parenteral, buccal and sublingual administration to patients,the daily dosage level of the compounds of formula (I) and theirpharmaceutically acceptable salts and solvates will be from 1 to 1000 mg(in single or divided doses). Thus tablets or capsules may contain from0.5 to 500 mg of active compound for administration singly, or two ormore at a time, as appropriate. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual patient and it will vary with the age, weight and response ofthe particular patient. The above dosages are exemplary of the averagecase; there can, of course, be individual instances where higher orlower dosage ranges are merited and such are within the scope of thisinvention.

Thus the invention provides a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,or a pharmaceutically acceptable solvate of either entity, together witha pharmaceutically acceptable diluent or carrier.

The invention also provides a compound of formula (I), or apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable solvate of either entity, or a pharmaceutical compositioncontaining any of the foregoing, for use as a medicament.

The invention further includes the use of a compound of formula (I), ora pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable solvate of either entity, or a pharmaceutical compositioncontaining any of the foregoing, for the manufacture of a medicament forthe curative or prophylactic treatment of deep vein thrombosis (DVT)after surgery, major medical illness, paralysis, malignancy, prolongedimmobilisation trauma, application of lower limb plaster casts, orfractures of the lower limbs or pelvis; recurrent DVT; DVT duringpregnancy when there is a previous history thereof; reocclusionfollowing thrombolytic therapy; chronic arterial obstruction; peripheralvascular disease; acute myocardial infarction; unstable angina; atrialfibrillation; thrombotic stroke; transient ischaemic attacks;disseminated intravascular coagulation; coagulation in extra-corporealcircuits; occlusion of arterio-venous shunts and blood vessel grafts(including coronary artery by-pass grafts); restenosis and occlusionfollowing angioplasty; neurodegenerative disorders; inflammatorydisorders; or scarring.

In a further aspect, the invention provides a method of treating amammal (including a human being) to cure or prevent deep vein thrombosis(DVT) after surgery, major medical illness, paralysis, malignancy,prolonged immobilisation trauma, application of lower limb plastercasts, or fractures of the lower limbs or pelvis; recurrent DVT; DVTduring pregnancy when there is a previous history thereof; reocclusionfollowing thrombolytic therapy; chronic arterial obstruction; peripheralvascular disease; acute myocardial infarction; unstable angina; atrialfibrillation; thrombotic stroke; transient ischaemic attacks;disseminated intravascular coagulation; coagulation in extra-corporealcircuits; occlusion of arterio-venous shunts and blood vessel grafts(including coronary artery bypass grafts); restenosis and occlusionfollowing angioplasty; neurodegenerative disorders; inflammatorydisorders; or scarring; which comprises treating said mammal with aneffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable solvate ofeither entity, or a pharmaceutical composition containing any of theforegoing.

The syntheses of the compounds of the invention and of the intermediatesfor use therein are illustrated by the following Examples andPreparations. The purity of the compounds (Rf) was routinely monitoredby thin layer chromatography using Merck Kieselgel 60 F₂₅₄ plates andthe following solvent systems (SS):

1. hexane:ether, 1:1;

2. ether;

3. dichloromethane:methanol:glacial acetic acid, 90:10:1;

4. hexane:ether, 4:1;

5. dichloromethane;

6. dichloromethane:methanol:0.880 aqueous ammonia, 95:5:0.5;

7. hexane:ethyl acetate, 3:7;

8. hexane:ethyl acetate, 1:1;

9. dichloromethane:methanol:0.880 aqueous ammonia, 90:10:1;

10. hexane:ethyl acetate, 3:1;

11. toluene:ethyl acetate, 4:1;

12. toluene:ethyl acetate, 1:1;

13. isobutyl methyl ketone:glacial acetic acid:water, 2:1:1 (upperphase);

14. ethyl acetate:ethanol, 4:1;

15. ethyl acetate:ethanol:glacial acetic acid, 90:10:0.4;

16. ethyl acetate:ethanol:glacial acetic acid, 80:20:1;

17. ethyl acetate:ethanol, 9:1;

18. hexane:ethyl acetate:diethylamine, 9:1:0.2;

19. hexane:ethyl acetate, 50:1;

20. dichloromethane:methanol, 97.5:2.5;

21. dichloromethane:ethanol. 97.5:2.5;

22. hexane:ethyl acetate, 1:4;

23. dichloromethane:methanol, 95:5;

24. dichloromethane:methanol:0.880 aqueous ammonia, 80:20:5;

25. dichloromethane:methanol:0.880 aqueous ammonia, 84:14:2;

26. ethyl acetate;

27. methanol:ethyl acetate:glacial acetic acid:0.880 aqueousammonia:water, 60:12:4:4:8;

28. dichloromethane:methanol:0.880 aqueous ammonia, 95:5:1;

29. hexane:ethyl acetate, 1:2.

¹ H Nuclear magnetic resonance (NMR) spectra were recorded using eithera Nicolet QE-300 or a Bruker AC-300 spectrometer and were in all casesconsistent with the proposed structures.

Mass spectra were obtained with a Fisons Instrument Trio 1000spectrometer using thermospray ionisation.

Room temperature means 20°-25° C.

EXAMPLE 1 4-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid dihydrochloride

1M Aqueous sodium hydroxide solution (12.5 ml, 12.5 mmol) was addeddropwise with stirring to an ice-cooled solution of the title compoundof Preparation 25 (1.57 g, 2.5 mmol) in water (10 ml) and the reactionmixture allowed to warm to room temperature. After 1.5 hours, thesolution was extracted with dichloromethane (2×15 ml) and then acidifiedto pH2 with 1M hydrochloric acid (13 ml). The solution was evaporated todryness under reduced pressure and residual traces of water removedazeotropically with 2-propanol. The dry residue was extracted withdichloromethane:2-propanol (1:1), then the combined extracts filteredand the filtrate evaporated under reduced pressure to yield the titlecompound (1.38 g, 90%) as a white powder. Rf 0.36 (SS 27). Found:C,48.46; H,6.11; N,12.24. C₂₆ H₃₆ N₆ O₅ S; 2HCl; H₂ O; 0.33 C₃ H₈ O;0.25 CH₂ Cl₂ requires C,48.35; H,6.43; N,12.41%.

EXAMPLE 2 4-Methyl-1-N-(3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid dihydrochloride

The title compound (0.6 g, 88%) was obtained from the title compound ofPreparation 33 (0.7 g, 1 mmol),using the procedure of Example 1, as awhite powder. Rf 0.27 (SS 27). Found: C,48.93; H,6.76; N, 11.84. C₂₇ H₃₈N₆ O₅ S; 2HCl; H₂ O; 0.33 C₃ H₈ O; 0.25 CH₂ Cl₂ requires C,49.10;H,6.58; N,12.16%. m/e 559.3 (M+H)⁺.

EXAMPLE 3 4(R)-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid dihydrochloride

The title compound (0.15 g, 100%) was obtained from the title compoundof Preparation 43 (0.16 g, 0.2 mmol), using the procedure of Example 1,as a white powder. Rf 0.33 (SS 27). Found: C,48.06; H,6.67; N,12.00. C₂₆H₃₈ N₆ O₅ S; 2HCl; H₂ O; 0.30 C₃ H₈ O; 0.25 CH₂ Cl₂ requires C,48.17;H,6.68; N,12.41 %. m/e 547.4 (M+H)⁺.

EXAMPLE 4 4-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid dihydrochloride

The title compound (496 mg, 84%) was obtained from the title compound ofPreparation 62 (576 mg, 0.91 mmol), using the procedure of Example 1, asa white powder. Rf 0.41 (SS27). Found: C,48.07; H,6.31; N,12.74. C₂₆ H₃₆N₆ O₅ S; 2HCl; H₂ O; 0.125 CH₂ Cl₂ requires C,48.55; H,6.28; N, 13.00%m/e 545 (M+H)⁺.

EXAMPLE 5 4(R)-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(S-carboxylicacid dihydrochloride

The title compound (300 mg, 79%) was obtained from the title compound ofPreparation 63 (360 mg, 0.57 mmol), using the procedure of Example 1, asa white powder. Rf 0.39 (SS27). Found: C,46.85; H,6.76; N,12.98. C₂₆ H₃₈N₆ O₅ S; 2HCl; 2H₂ O; 0.15 CH₂ Cl₂ requires C,46.99; H,6.68; N,1257%.

EXAMPLE 6 4(S)-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid dihydrochloride

The title compound (270 mg, 92%) was obtained from the title compound ofPreparation 64 (330 mg, 0.48 mmol), using the procedure of Example 1, asa white powder. Rf 0.37 (SS 27). Found: C,46.76; H,6.54; N,12.39. C₂₆H₃₈ N₆ O₅ S; 2HCl; 1.50 H₂ O; 0.35 CH₂ Cl₂ requires C,46.79; H,6.51;N,12.43%.

PREPARATION 12-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl chloride

(a) 2-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline

This intermediate was obtained by the method described in J. Med. Chem.,1980, 23, 837 and used directly in step (b).

(b) The title compound was also obtained by the method described in J.Med. Chem., 1980, 23, 837, using the intermediate from (a) above, as awhite solid (52.9% yield based on 1,2,3,4-tetrahydroisoquinoline), m.p.104°-105° C., after crystallisation from ether. Rf 0.25 (SS 1).

PREPARATION 22-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-6-sulphonyl chloride

Crystallisation of material recovered from the mother-liquors ofPreparation 1(b), from diisopropyl ether, afforded the title compound(3.6% yield based on 1,2,3,4-tetrahydroisoquinoline), m.p. 110°-112° C.Rf 0.36 (SS 1).

PREPARATION 33-Trifluoroacetyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonylchloride

(a) 2,3,4,5-Tetrahydro-1H-3-benzazepine

This starting material was obtained by the method described in Helv.Chim. Acta, 1935, 18, 1388.

(b) 3-Trifluoroacetyl-2,3,4,5-tetrahydro-1H-3-benzazepine

Trifluoroacetic anhydride (6.0 g, 28.56 mmol) was added dropwise over 20minutes to a stirred, ice-cooled solution of the material from (a) above(4.2 g, 28.56 mmol) and N-methylmorpholine (2.89 g, 28.56 mmol) indichloromethane (45 ml). After 2.5 hours at room temperature, thereaction solution was washed successively with water, 1M aqueous citricacid solution and water, dried (MgSO₄) and evaporated under reducedpressure to yield a yellow solid, which was triturated with hot hexane.Filtration, concentration and cooling of the combined hexane solutionsafforded the required product (5.96 g) as a pale yellow solid, m.p.78°-80° C. Found: C,58.85; H,4.93; N,5.75. C₁₂ H₁₂ F₃ NO requiresC,59.25; H,4.97; N,5.76%.

(c) Chlorosulphonic acid (10.4 ml, 0.156 mol) was added dropwise to astirred, cold solution of the product from (b) above (5.85 g, 11.7 mmol)in dichloromethane, whilst ensuring that the temperature of the reactionmixture was held between -12° and -8° C. After 2 days at roomtemperature, the reaction solution was poured onto ice and the aqueousphase separated and extracted with dichloromethane. The combined organicextracts were dried (MgSO₄) and evaporated under reduced pressure toprovide an oil (7.9 g) which was purified by chromatography on silicagel, using a mixture of hexane and ether (1:3) as eluant, to give thetitle compound as a colourless oil which eventually solidified.Crystallisation of a sample from diisopropyl ether produced a whitesolid. m.p. 87°-88° C. Found: C,41.75; H,3.18; N,3.92. C₁₂ H₁₁ Cl F₃ NO₃S requires C,42.17; H,3.24; N,4.10%.

PREPARATION 41(R,S)-Methoxymethyl-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonylchloride

(a) N-Methoxyacetyl-2-phenylethylamine

Methoxyacetyl chloride (19.57 g, 0.18 mol) was added over 10 minutes toa stirred, ice-cooled solution of 2-phenylethylamine (21.85 g, 0.18 mol)and N-ethyldiisopropylamine (23.26 g, 0.18 mol) in dichloromethane (200ml). After 2 hours at room temperature, the solvent was removed underreduced pressure and the residue partitioned between ether and water.The organic phase was washed successively with 1M aqueous citric acidsolution, water, saturated aqueous sodium bicarbonate solution andwater, dried (MgSO₄) and evaporated under reduced pressure to providethe required product (29.65 g) as an oil, Rf 0.32 (SS 2), which was usedwithout further purification in the next step.

(b) 1-Methoxymethyl-3,4-dihydroisoquinoline

Phosphorous pentoxide (25 g, 0.176 mol) was added to a stirred solutionof the product from (a) above (14.47 g, 0.075 mol) in xylene (35 ml) andthe resulting mixture heated under reflux for 2.5 hours. The solvent wasdecanted from the resulting black gum which was triturated with xyleneand then, when cool, with ether. Water was then carefully added, withice-cooling, and the resulting mixture basified with 2M aqueous sodiumhydroxide solution and then extracted with ether. The extract was washedwith water, dried (MgSO₄) and evaporated under reduced pressure to givea brown oil (13.4 g) which was purified by chromatography on silica gel,using ether as eluant, to afford the required compound (5.88 g) as anorange oil. Rf 0.28 (SS 2).

(c) 1(R,S)-Methoxymethyl-1,2,3,4-tetrahydroisoquinoline

Sodium triacetoxyborohydride (8.11 g, 38.3 mmol) was added to a stirred,ice-cooled solution of the product from (b) above (6.1 g, 34.8 mmol) inmethanol (80 ml), then the resulting mixture stirred for 18 hours atroom temperature before being quenched with water. The bulk of thesolvent was removed under reduced pressure, then the residue basifiedwith 1M aqueous sodium hydroxide solution and extracted withdichloromethane. The extract was washed with saturated brine, dried(MgSO₄) and evaporated under reduced pressure to furnish the requiredproduct (6.19 g) as an orange oil, Rf 0.25 (SS 3), m/e 178 (M+H)⁺, whichwas used without further purification in the next step.

(d)1(R,S)-Methoxymethyl-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline

Trifluoroacetic anhydride (10.26 ml, 72.6 mmol) was added dropwise over0.5 hour to a stirred, ice-cooled solution of the product from (c) above(12.26 g, 69.17 mmol) and N-methylmorpholine (7.35 g, 72.6 mmol) indichloromethane (150 ml). After 2 hours at room temperature, the solventwas removed under reduced pressure and the residue partitioned betweenether and water. The organic phase was washed successively with waterbasified to pH ca. 8 with sodium bicarbonate, 1M aqueous citric acidsolution and water, dried (MgSO₄) and evaporated under reduced pressureto provide an oil (19.45 g) which was purified by chromatography onsilica gel, using a mixture of hexane and ether (4:1) as eluant, to givethe required compound (16.16 g) as a clear oil. Rf 0.27 (SS 4). Found:C,56.83; H,5.19; N,5.02. C₁₃ H₁₄ F₃ NO₂ requires C,57.14; H,5.16;N,5.12%.

(e) The title compound was obtained from the product of (d) above, usingthe method of Preparation 3(c), as a pale yellow oil which solidifiedwhen chilled. Rf 0.35 (SS 5).

PREPARATION 51(R,S)-(N,N-Dimethylcarbamoylmethyl)-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonylchloride

(a)1(R,S)-(N,N-Dimethylcarbamoylmethyl)-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline

1-Ethoxycarbonylmethyl-1,2,3,4-tetrahydroisoquinoline (13.15 g, 60mmol), obtained by the method described in J. Org. Chem., 1965, 30,3667, was dissolved in a 30% solution of dimethylamine in ethanol (100ml). Dimethylamine (32 g) was added and the reaction mixture heated in asteel bomb at 120° C. for 24 hours and at 150° C. for a further 24hours, then evaporated under reduced pressure to provide the requiredcrude amide (12.9 g) as an oil, Rf 0.13 (SS 3) plus trace of esterstarting material at Rf 0.33, which was used without furtherpurification in the next step.

Trifluoroacetic anhydride (12.12 g, 72 mmol) was added dropwise over 0.5hour to a stirred, ice-cooled solution of the crude amide (12.9 g) andN-ethyidiisopropylamine (10.34 g, 80 mmol) in dichloromethane (120 ml).After 3 hours at room temperature, the solvent was removed under reducedpressure and the residue partitioned between ether and water. Theorganic phase was washed successively with water, 5% aqueous sodiumbicarbonate solution, 1M aqueous citric acid solution and water, dried(MgSO₄) and evaporated under reduced pressure to give an orange oilwhich was purified by chromatography on silica gel, using a mixture ofhexane and ethyl acetate (3:7) as eluant, to furnish the requiredcompound (14.3 g). Rf 0.35 (SS 7). Found: C,56.88; H,5.38; N,8.85. C₁₅H₁₇ F₃ N₂ O₂ requires C,57.32; H,5.45; N,8.91%.

(b) The title compound (84% yield) was obtained from the product of (a)above, using the procedure of Preparation 3(c), as a white foam. Rf 0.47(SS 7).

PREPARATION 65-Methyl-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-8-sulphonylchloride

(a) 5-Methylisoquinoline

A 3M solution of methyl magnesium iodide in ether (50 ml, 0.15 mol) wasadded dropwise to a stirred, ice-cooled solution of 5-bromoisoquinoline(21 g, 0.10 mol), obtained by the method described in J. Org. Chem.,1964, 29, 329, and 1,3-bis(diphenylphosphino)propane!nickel(II) chloride(400 mg, 0.7 mmol) in anhydrous ether and the reaction mixture heatedunder reflux for 5 days, allowed to cool, then poured into water (500ml). The organic phase was separated, combined with ether extracts ofthe aqueous phase, washed with saturated brine, dried (MgSO₄) andevaporated under reduced pressure to yield an oil, chromatography ofwhich on silica gel, using a 5-50% ether in hexane elution gradient,provided the required product (8.4 g). Rf 0.40 (SS 8), m/e 144 (M+H)⁺.Found: C,79.57; H,6.26; N,8.61. C₁₀ H₉ N; 0.27 C₄ H₈ O₂ requiresC,79.70; H,6.74; N,8.39%.

However, the major component of the chromatographic purificationprocedure was a mixture (18.4 g) of product and starting material which,on crystallisation from hexane, afforded a 2:1 mixture (14.8 g) of5-bromoisoquinoline and 5-methylisoquinoline.

(b) A stirred, ice-cooled solution of the above 2:1 mixture (14.3 g) indichloromethane (150 ml) was saturated with hydrogen chloride and thenevaporated under reduced pressure to afford the correspondinghydrochloride salt which was collected and dried. A stirred mixture ofplatinum oxide (1 g) and a solution of the preceding hydrochloride saltin ethanol (150 ml) was hydrogenated for 30 hours at 50 psi (3.45 bar)and room temperature, then filtered. The filtrate was evaporated underreduced pressure and the residue chromatographed on silica gel, using amixture of dichloromethane:methanol: 0.880 aqueous ammonia solution(90:10:1) as eluant, to give an 85:15 mixture (5.62 g) of5-methyl-1,2,3,4-tetrahydroisoquinoline and5-bromo-1,2,3,4-tetrahydroisoquinoline as an oil; major component: Rf0.32 (SS 9), m/e 148 (M+H)⁺.

The above 85:15 mixture was converted to the corresponding2-trifluoroacetyl derivative mixture, using the procedure described inPreparation 3(b), to afford an oil; major component: Rf 0.90 (SS 10),m/e 244 (M+H)⁺.

The above crude mixture containing 85% of5-methyl-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline, waschlorosulphonated following the procedure described in Preparation 3(c)to provide a yellow solid which, on purification by chromatography onsilica gel using an elution gradient of 10-50% ethyl acetate in hexane,gave the title compound as a white solid, m.p. 152°-153° C. Found:C,42.18; H,3.24; N,4.10. C₁₂ H₁₁ Cl F₃ NO₃ S requires C,42.12; H,3.10;N,3.85%.

PREPARATION 7 N-t-Butoxycarbonyl-3-(3-pyridyl)-(S)-alanine

Anhydrous potassium carbonate (7.5 g, 54 mmol) was added to a stirred,ice-cooled suspension of 3-(3-pyridyl)-(S)-alanine (Int. J. Pept. Prot.Res.; 1987, 29, 118; 9 g, 54 mmol) in water (50 ml), followed by thedropwise addition of a solution of di-t-butyl dicarbonate (18 g, 82mmol) in 1,4-dioxan (25 ml) over 10 minutes. The resulting mixture wasallowed to warm to room temperature and stir for 18 hours. The reactionmixture was concentrated by evaporation of the bulk of the 1,4-dioxanunder reduced pressure and extracted with ethyl acetate (2×20 ml). ThepH of the aqueous solution was adjusted to ca. 3 with solid citric acid,solid sodium chloride added to saturation and extraction with ethylacetate (5×20 ml) effected. The combined organic extracts were dried(MgSO₄), filtered and evaporated to low bulk under reduced pressure,whereupon the product started to crystallise. After chilling at 0° C.for 1 hour, the crystals were collected, washed with ether and dried togive the required product (10.36 g, 72%). Rf 0.34 (SS 13).

α!_(D) ²⁵ +10° (c=0.1, CH₃ OH). Found: C,58.69; H,7.17; N,10.50. C₁₃ H₁₈N₂ O₄ requires C,58.63; H,6.81; N,10.52%.

PREPARATION 8 N-t-Butoxycarbonyl-3-(3-pyridyl)-(S)-alanine t-butyl ester

To a stirred, ice-cooled solution of the title compound of Preparation 7(10 g, 37.5 mmol) and t-butanol (14 g, 189 mmol) in dichloromethane (100ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (10 g, 52 mmol) and 4-dimethylaminopyridine (2.3 g, 18.8mmol). The reaction mixture was allowed to warm to room temperature andstir for 18 hours. The solvent was evaporated under reduced pressure andthe residue dissolved in ethyl acetate (100 ml). This solution waswashed with water (2×30 ml) and brine (20 ml), dried (MgSO₄), filteredand evaporated under reduced pressure to yield an oil, which waspurified by chromatography on silica gel using ethyl acetate:hexane(1:1) as eluant to afford the title compound (12.2 g, 100%) as a clearoil. Rf 0.59 (SS 26). m/e 322.9 (M+H)⁺.

PREPARATION 9 N-t-Butoxycarbonyl-3-(1-benzyl-3-pyridinium)-(S)-alaninet-butyl ester bromide

To a stirred solution of the title compound of Preparation 8 (1 g, 3.1mmol) in dry ethanol (10 ml) at room temperature was added a solution ofbenzyl bromide (0.79 g, 4.6 mmol) in dry ethanol (2 ml), dropwise, over5 minutes. After 18 hours the ethanol was evaporated under reducedpressure. The residue was dissolved in acetonitrile (10 ml) and thesolution was extracted with hexane (3×5 ml) and then evaporated todryness under reduced pressure. Solvent traces were removedazeotropically with dichloromethane to furnish the required product(1.23 g, 96%) as a white foam. Rf 0.49 (SS 13). Found: C,56.40; H,7.07;N,5.10. C₂₃ H₃₃ BrN₂ O₄ ; 0.17 CH₂ Cl₂ requires C,56.14; H,6.77;N,5.65%.

PREPARATION 10N-t-Butoxycarbonyl-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alaninet-butyl ester

Sodium borohydride (0.19 g, 5 mmol) was added in two equal portions over5 minutes to a stirred, ice-cooled solution of the title compound ofPreparation 9 (0.7 g, 1.7 mmol) in ethanol (10 ml). After 1.5 hours, thesolution was evaporated to dryness under reduced pressure. The residuewas dissolved in dichloromethane (20 ml) and the solution was washedwith saturated aqueous sodium bicarbonate solution and brine, dried(MgSO₄), filtered and evaporated under reduced pressure. The residue waspurified by chromatography on silica gel, using an elution gradient ofethyl acetate:hexane (0:100 to 20:80), to afford the title compound(0.36 g, 91%) as an oil. Rf 0.42 (SS 8). m/e 417.3 (M+H)⁺.

PREPARATION 11 3-(1-Benzyl-1 2,5,6-tetrahydro-3-pyridyl)-(S)-alaninet-butyl ester

The title compound of Preparation 10 (1 g, 2.4 mmol) was dissolved in96% formic acid (10 ml) and the solution was stirred at room temperaturefor 4 hours. The formic acid was then removed under reduced pressure at≦30° C. The residue was dissolved in 1M aqueous citric acid solution (20ml) and the solution extracted with ethyl acetate (2×10 ml). The aqueousphase was basified to pH8 with solid sodium bicarbonate and extractedwith dichloromethane (3×10 ml). The combined organic extracts were dried(MgSO₄), filtered and evaporated under reduced pressure to yield therequired product (0.74 g, 97%) as an oil, which was used without furtherpurification. Rf 0.73 (SS 9).

PREPARATION 12N-(2-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alaninet-butyl ester

2-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl chloride(Preparation 1; 2.26 g, 6.9 mmol) was added dropwise over 5 minutes as asolution in dichloromethane (5 ml) to a stirred, ice-cooled solution ofthe title compound of Preparation 11 (1.82 g, 5.7 mmol) andN-ethyidiisopropylamine (1.1 g, 8.5 mmol) in dichloromethane (10 ml).The resulting solution was allowed to warm to room temperature and stirfor 18 hours. The solvent was evaporated under reduced pressure and theresidue dissolved in ethyl acetate (30 ml). This solution was washedwith water (10 ml), saturated aqueous sodium bicarbonate solution (2×10ml) and saturated brine (10 ml), dried (MgSO₄), filtered and evaporatedunder reduced pressure. The residue was purified by chromatography onsilica gel, using an elution gradient of ethyl acetate:hexane (10:90 to50:50), to furnish the required product (3 g, 86%) as a white foam. Rf0.63 (SS 26). Found: C,58.70; H,5.75; N,6.64. C₃₀ H₃₆ F₃ N₃ O₅ S; 0.10CH₂ Cl₂ requires C,58.66; H,5.92; N,6.82%. m/e 608.4 (M+H)⁺.

PREPARATION 13N-(2-Trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl-(S)-alaninehydrochloride

A stirred, ice-cooled solution of the title compound of Preparation 12(3 g, 4.9 mmol) in dichloromethane (30 ml) was saturated with hydrogenchloride and then allowed to attain room temperature. After 6 hours thesolvent and excess hydrogen chloride were removed under reducedpressure. Residual traces of hydrogen chloride were removedazeotropically with dichloromethane to yield the title compound (3 g,100%) as a white foam. Rf 0.29 (SS 13). Found: C,50.40; H,4.90; N,6.73.C₂₆ H₂₈ F₃ N₃ O₅ S; HCl; 0.50 CH₂ Cl₂ requires C,50.47; H,4.79; N,6.66%.m/e 552.4 (M+H)⁺.

PREPARATION 14 4(R)-Methylpiperidine-2(R)-carboxylic acid ethyl ester

The title compound was obtained by the method described in Biochem.Biophys. Res. Comm., 1981, 101, 440.

PREPARATION 15 4-Methyl-1,2,3,6-tetrahydropyridine-2(R)-carboxylic acidethyl ester hydrochloride

(a) 4-Methyl-1-1-(S)-phenylethyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylic acid ethylester

This intermediate was obtained by the method described inTetrahedron:Asymmetry, 1991 2, 1263.

(b) A stirred solution of the above intermediate (13.56 g, 49.7 mmol) intoluene (150 ml), under nitrogen, was heated under reflux for 2 hours,using a Dean-Stark trap. 1,8-Bis(dimethylamino)naphthalene (1.08 g, 5.0mmol) was then added, heating continued for a further 1 hour, thereaction mixture allowed to cool and the Dean-Stark trap removed. Afterthe addition of 1-chloroethyl chloroformate (10.7 ml, 14.2 g, 99.5mmol), the reaction mixture was stirred under reflux for 16 hours,allowed to cool, treated with absolute ethanol (80 ml), stirred underreflux for 2 hours more and evaporated under reduced pressure. Theresidue was partitioned between ethyl acetate (100 ml) and 1Mhydrochloric acid (50 ml), then the aqueous phase separated, neutralisedwith solid sodium bicarbonate and extracted with dichloromethane (3×200ml). Evaporation under reduced pressure of the dried (MgSO₄), combinedextracts provided a brown residue which was purified by chromatographyon silica gel, using a 0-5% methanol in dichloromethane elutiongradient, and then converted to the required hydrochloride salt usingexcess ethereal hydrogen chloride. The product was further purified bydissolution in dichloromethane (20 ml), filtration (to remove residualsilica gel), dilution of the filtrate with ether (200 ml), filtration,washing of the precipitate with ether and drying in vacuo. A sample ofthis purified product (5.77 g) was crystallised from a mixture of etherand ethanol to afford the title compound as white crystals, m.p.110°-111° C. Rf 0.35 (SS 3).

α!_(D) ²⁵ +113.7° (c=1.0, CH₃ CH₂ OH). Found: C,52.79; H,7.94; N,6.68.C₉ H₁₅ NO₂ ; HCl requires C,52.55; H,7.84; N,6.81%.

PREPARATION 16 4-Methyl-1,2,3,6-tetrahydropyridine-2(R)-carboxylic acidmethyl ester p-toluenesulphonate

(a) 4-Methyl-1-1-(S)-phenylethyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylic acidmethyl ester

This intermediate was obtained by the method described in Tetrahedron,1992, 48, 9707 as the major diastereoisomer of a mixture also containingthe 1(S), 2(S)-diastereoisomer (ratio 3.7:1 by NMR spectroscopy).

Formation of the hydrochloride salt using hydrogen chloride in toluene.followed by two crystallisations from ethyl acetate and liberation ofthe free base, afforded the required 1(S),2(R)-diastereoisomer insufficiently stereochemically pure form (S,R:S,S>97:3) for furtherprocessing.

(b) N-Deprotection of the previous product was effected by themethodology described in J. Org. Chem., 1984, 49, 2081, (see alsoPreparation 21), using 1-chloroethyl chloroformate as the"debenzylation" reagent, followed by conversion of the resultinghydrochloride salt to the corresponding p-toluenesulphonate, m.p.141°-143° C. Rf 0.61 (SS 9).

PREPARATION 17 4-Methyl-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylicacid ethyl ester hydrochloride

(a) 1-Benzyl-4-methyl-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylic acidethyl ester

This intermediate was obtained by the method described in TetrahedronAsymmetry, 1991, 2, 1263.

(b) The title compound (82% yield) was obtained from the intermediate of(a) above, using the method of Preparation 15(b), as a white solid, m.p.130°-130.5° C. Rf 0.35 (SS 3).

PREPARATION 18 4-Methyl-1,2,3,6-tetrahydropyridine-2(S)-carboxylic acidethyl ester hydrochloride

(a) 1-Benzyl-4-methyl-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylic acidhydrochloride

A stirred solution of1-benzyl-4-methyl-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylic acidethyl ester (Preparation17(a); 20.1 g, 77.5 mmol) in 5M hydrochloricacid (200 ml) was heated at 100° C. for 4.5 hours and then evaporated todryness under reduced pressure. Residual water was removedazeotropically using dichloromethane followed by toluene to give therequired product (24.0 g) as a white foam, Rf 0.40 (SS 13), which wasused without further purification in the next step.

(b) 1-Benzyl-4-methyl-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylic acidN-acetyl-(1R,2S)-ephedrine ester

1,3-Dicyclohexylcarbodiimide (20.6 g, 0.1 mol) was added to a stirredsolution of the product from (a) above (24.0 g, 89.6 mmol),N-acetyl-(1R,2S)-ephedrine (18.47 g, 89.2 mmol), obtained by the methoddescribed in J. Amer. Pharmaceut. Assoc., 1952, 41, 545, (see J. Med.Chem., 1965, 8, 466), N-ethyldiisopropylamine (12.9 g, 0.1 mmol) and4-dimethylaminopyridine (9.51 g, 77.5 mmol) in dichloromethane (250 ml).After 3 days at room temperature, the reaction mixture was filtered andthe filtrate evaporated to dryness under reduced pressure. The residuewas partitioned between ethyl acetate and water, then the organic phaseseparated, washed with water, dried (MgSO₄) and evaporated under reducedpressure to give the crude product, which was purified by chromatographyon silica gel, using ether as eluant, to furnish the required pureproduct (25.5 g) as an oil. Rf 0.35 (SS 2). Found: C,73.34; H,7.71;N,5.73. C₂₆ H₃₂ N₂ O₃ ; 0.30 C₄ H₁₀ O requires C,73.78; H,7.91; N,6.3%.

(c)4-Methyl-1-(2,2,2-trichloroethoxycarbonyl)-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylicacid N-acetyl-(1R,2S)-ephedrine ester

Sodium bicarbonate (11.21 g, 133 mmol), then 2,2,2-trichloroethylchloroformate (11.7 ml, 17.97 g, 84.8 mmol), were added to a stirredsolution of the product from (b) above (25.48 g, 60.6 mmol) in drydichloromethane (200 ml) and the resulting mixture was heated underreflux for 22 hours. The solvent was removed under reduced pressure andthe residue partitioned between ethyl acetate and water. The separatedorganic phase was washed with saturated brine, dried (MgSO₄) andevaporated under reduced pressure, then the residue purified bychromatography on silica rel, using ether as eluant, to afford therequired product (28.1 g) as a gum. Rf 0.40 (SS 2). Found: C,52.10;H,5.38; N,5.24. C₂₂ H₂₇ Cl₃ N₂ O₅ requires C,52.24; H,5.38; N,5.54%.

(d) 4-Methyl-1,2,3,6-tetrahydropyridine-2(R)-carboxylic acidN-acetyl-(1R,2S)-ephedrine ester and4-methyl-1,2,3,6-tetrahydropyridine-2(S)-carboxylic acidN-acetyl-(1R,2S)-ephedrine ester

1M Aqueous potassium dihydrogen phosphate solution (40 ml, 40 mmol),then zinc dust (40 g, 610 mmol), were added to a rapidly stirredsolution of the product from (c) above (32.9 g, 65 mmol) intetrahydrofuran (200 ml). After 1 hour at room temperature, the reactionmixture was filtered and the bulk of the organic solvent removed underreduced pressure, then the residue basified with saturated aqueoussodium bicarbonate solution and extracted with ethyl acetate. Theextract was washed with saturated brine, dried (MgSO₄) and evaporatedunder reduced pressure to give an oil (21.46 g), which waschromatographed on silica gel using an elution gradient of ethanol:ethylacetate (1:4 to 3:7), to provide firstly the 2(R)-diastereoisomericester (2.75 g), Rf 0.30 (SS 14), m/e 331 (M+H)⁺, followed by the2(S)-diastereoisomeric ester (2.90 g), Rf 0.22 (SS 14), m/e 331 (M+H)⁺,each as a pale yellow oil.

(e)N-t-Butoxycarbonyl-4-methyl-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid N-acetyl-(1R,2S)-ephedrine ester

Di-t-butyl dicarbonate (4.4 g, 20.2 mmol) was added to a stirredsolution of the 2(S)-ester product from (d) above (5.13 g, 15.5 mmol)and N-methylmorpholine (2.04 g, 20.2 mmol) in dichloromethane (40 ml).After 5 hours at room temperature, more di-t-butyl dicarbonate (1.35 g,6.2 mmol) was added and the reaction mixture stirred for a further 15hours before removal of the solvent under reduced pressure. The residualmixture was partitioned between ethyl acetate and water, then theorganic phase washed successively with 1M aqueous citric acid solution,saturated aqueous sodium bicarbonate solution and saturated brine, dried(MgSO₄) and evaporated under reduced pressure to give a gum which waspurified by chromatography on silica gel, using ether as eluant, toafford the required product (6.02 g) as a gum. Rf 0.45 (SS 2). Found:C,66.84; H,7.97; N,6.50. C₂₄ H₃₄ N₂ O₅ requires C,66.95; H,7.96;N,6.51%.

(f) N-t-Butoxycarbonyl-4-methyl-1,2,3,6-tetrahydropyridine-2(S)-carboxylic acid

1M Aqueous sodium hydroxide solution (69 ml, 69 mmol) was added to astirred solution of the product from (e) above (5.97 g, 13.9 mmol) in1,4-dioxan (60 ml). After 3 hours at room temperature, solid citric acid(5.53 g, 26 mmol) was added and the bulk of the solvent removed underreduced pressure. The residual suspension was basified to pH 10 with 1Maqueous sodium hydroxide solution, washed with dichloromethane,acidified to pH 3 with solid citric acid and extracted with ethylacetate. The organic extract was washed with saturated brine, dried(MgSO₄) and evaporated under reduced pressure to provide the requiredproduct (3.47 g) as a gum. Rf 0.60 (SS 17).

(g)N-t-Butoxycarbonyl-4-methyl-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid ethyl ester

1,3-Dicyclohexylcarbodiimide (4.3 g, 20.8 mmol) was added to a stirredsolution of the product from (f) above (3.35 g, 13.9 mmol), ethanol (4.1ml, 69.4 mmol) and 4-dimethylaminopyridine (1.7 g, 13.9 mmol) indichloromethane (40 ml). After 18 hours at room temperature, glacialacetic acid (0.4 ml) was added and the reaction mixture stirred for afurther 0.5 hour before being filtered. The residue obtained byevaporation of the filtrate under reduced pressure was partitionedbetween ether and water, then the organic phase washed successively with1M aqueous citric acid solution, saturated aqueous sodium bicarbonatesolution and saturated brine, dried (MgSO₄) and evaporated under reducedpressure to give an oil (5.15 g), which was purified by chromatographyon silica gel, using hexane:ether (4:1) as eluant, to furnish therequired product (3.4 g) as a clear oil. Rf 0.30 (SS 4). m/e 270 (M+H)⁺.

(h) A stirred, ice-cooled solution of the product from (g) above (3.13g, 11.62 mmol) in ethyl acetate (30 ml) was satured with hydrogenchloride over 0.5 hour and then stood at room temperature for a further2 hours. The solvent was evaporated under reduced pressure and theresidue crystallised from a mixture of ether and ethanol to afford thetitle compound (2.20 g) as white crystals, m.p. 108°-109° C. Rf 0.35 (SS3),

α!_(D) ²⁵ -106.5° C. (c=1.0, CH₃ CH₂ OH). Found: C,52.49; H,7.90;N,6.70. C₉ H₁₅ NO₂ ; HCl requires C,52.55; H,7.84; N,6.81%.

The compound obtained by carrying out steps (e), (f), (g) and (h) on the2(R)-ester from (d) above was found to be identical with the titlecompound of Preparation 15.

PREPARATION 19 4-Ethyl-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylic acidethyl ester hydrochloride

(a) 2-Cyano-4-ethyl-1-methyl-1,2,3,6-tetrahydropyridine hydrochloride

Methyl iodide (74.7 ml, 170.33 g, 1.22 mol) was cautiously addedportionwise to a stirred solution of 4-ethylpyridine (107.2 g, 1 mol) inacetone (500 ml), the resulting mixture heated under reflux for 2 hoursand allowed to cool, then a portion of solvent (ca. 100 ml) removedunder reduced pressure. Addition of ether (1.0 l), collection andwashing with ether of the precipitate, followed by drying in vacuo,provided the required quaternary iodide (245 g) as a very hygroscopicsolid. 6M Hydrochloric acid (130 ml) was slowly added to a stirredsolution of potassium cyanide (130 g, 2.5 mol) in water (260 ml) coveredby a layer of ether (400 ml), ensuring that the temperature wasmaintained below 15° C. Successive, portionwise addition of the abovequaternary salt (139.49 g, 0.56 mol) and sodium borohydride (27 g, 0.71mol) over 15 minutes gave a milky mixture, which was stirred at ca. 10°C. for 0.5 hour and then at room temperature for a further 4 hours. Theether phase was removed by suction and combined with an ether extract ofthe aqueous phase, then washing with saturated brine and drying (MgSO₄)effected. The ether solution was ice-cooled, with stirring, and methyliodide (6 ml) added to precipitate any unwanted4-ethyl-1-methylpiperidine as the derived quaternary iodide. Filtration,followed by treatment of the filtrate with excess 1M ethereal hydrogenchloride, afforded the required product (43.77 g) as an oil, Rf 0.20 (SS18), which was used without further purification in the next step.

(b)4-Ethyl-1-(2,2,2-trichloroethoxycarbonyl)-1,2,3,6-tetrahydropyridine-2(R,S)-carboxylicacid ethyl ester

A stirred solution of the product from (a) above (43.5 g, 0.23 mol) inethanol (100 ml) was saturated with hydrogen chloride, heated underreflux for 6.5 hours and then evaporated to dryness under reducedpressure. The residue was basified with aqueous sodium carbonatesolution, then extraction with ethyl acetate effected. The extract waswashed with saturated brine, dried (MgSO₄) and evaporated under reducedpressure to give the crude ethyl ester, which was partially purified bychromatography on silica gel, using hexane:ethyl acetate:diethylamine(90:5:2) as eluant, to provide an oil (8.0 g).

Sodium carbonate (10.0 g, 94 mmol) and 2,2,2-trichloroethylchloroformate (13.0 g, 61 mmol) were added successively to a stirredsolution of the above crude ester (8.0 g, 40 mmol) in dichloromethane(200 ml) and the resulting mixture heated under reflux for 20 hours,then evaporated under reduced pressure. The residue was partitionedbetween ethyl acetate and water, then the organic phase dried (MgSO₄)and evaporated under reduced pressure to give an oil which was purifiedby chromatography on silica gel, using hexane:ethyl acetate (50:1) aseluant, to furnish the required product (4.8 g) as a colourless oil. Rf0.25 (SS 19).

(c) 1M Aqueous potassium dihydrogen phosphate solution (70 ml, 70 mmol),then zinc dust (46 g, 700 mmol), were added to a rapidly stirredsolution of the product from (b) above (4.76 g, 13.3 mmol) intetrahydrofuran (220 ml). After 2 hours at room temperature more zincdust (5 g, 76 mmol) was added and the reaction mixture stirred for afurther hour before being filtered. The filter pad was washed with waterand tetrahydrofuran, then the bulk of the organic solvent removed fromthe combined filtrate and washings. The residual mixture was acidifiedwith 2M hydrochloric acid (30 ml), washed with ethyl acetate to removestarting material (2.32 g recovered), neutralised with solid potassiumcarbonate and extracted with dichloromethane. The combined organicextracts were washed with saturated brine, dried (MgSO₄), treated withexcess ethereal hydrogen chloride and evaporated under reduced pressure.Purification of the residue by chromatography on silica gel, usingdichloromethane:methanol (95:5) as eluant, furnished the title compound(330 mg), as a colourless waxy solid. m/e 184 (M+H)⁺.

PREPARATION 20 4-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

Dimethylformamide (0.25 ml) was added in one portion to a stirred,ice-cooled solution of the title compound of Preparation 13 (3 g, 5.1mmol) and oxalyl chloride (2.6 g, 20.5 mmol) in dichloromethane (30 ml).After 2 hours at room temperature the solvent and excess oxalyl chloridewere removed by evaporation under reduced pressure. Residual traces ofoxalyl chloride were removed azeotropically with dichloromethane to givethe crude acyl chloride as a foam, which was then dissolved indichloromethane (30 ml). To this stirred, ice-cooled solution wereadded, successively, the title compound of Preparation 16 (1.83 g, 5.6mmol) and N-ethyidiisopropylamine (2.3 g, 17.8 mmol). The resultingsolution was stirred at room temperature for 18 hours and thenevaporated under reduced pressure. The residue was dissolved in ethylacetate (50 ml) and the solution washed successively with water,saturated aqueous sodium bicarbonate solution and saturated brine, dried(MgSO₄) and evaporated under reduced pressure to give the crude product,which was purified by chromatography on silica gel using an elutiongradient of ethyl acetate:hexane (10:90 to 100:0) to afford the titlecompound (3 g, 85%) as a white foam. Rf 0.32 (SS 26). Found C,59.08;H,5.27; N,7.62. C₃₄ H₃₉ F₃ N₄ O₆ S requires C,59.29; H,5.71; N,8.13%.m/e 689.1 (M+H)⁺.

PREPARATION 21 4-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

1-Chloroethyl chloroformate (0.93 g, 6.5 mmol) was added dropwise as asolution in dichloromethane (3 ml) to a stirred, ice-cooled solution ofthe title compound of Preparation 20 (3 g, 4.3 mmol) and1,8-bis(dimethylamino)naphthalene (0.09 g, 0.4 mmol) in dichloromethane(12 ml). The resulting solution was allowed to warm to room temperature.After 3 hours the reaction mixture was washed with 1M aqueous citricacid solution (2×5 ml) and saturated brine (5 ml), dried (MgSO₄),filtered and evaporated under reduced pressure. The residue wasdissolved in methanol (25 ml) and the solution heated to reflux for 45minutes. The solvent was evaporated under reduced pressure and the crudeproduct purified by chromatography on silica gel, using an elutiongradient of dichloromethane:hexane (75:25 to 100:0), followed bydichloromethane:methanol:0.880 aqueous ammonia (99:1:0.5 to 95:5:0.5),to furnish the required product (2 g, 76%) as a white foam. Rf 0.24 (SS9). Found: C,52.86; H,5.38; N,8.65. C₂₇ H₃₃ F₃ N₄ O₆ S; 0.20 CH₂ Cl₂requires C,53.06; H,5.46; N,9.10%. m/e 599 (M+H)⁺.

PREPARATION 22 4-Methyl-1-{N-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

A stirred, ice-cooled solution of the title compound of Preparation 21(2 g, 3.3 mmol) in dichloromethane (20 ml) was treated sequentially withtriethylamine (1 g, 9.9 mmol), bis t-butoxycarbonyl-S-methylisothiourea(J. Med. Chem., 1993, 36, 2956; 1.05 g, 3.6 mmol) and mercuric chloride(1 g, 3.7 mmol) and then allowed to warm to room temperature and stirfor 18 hours. The reaction mixture was filtered and the filtrate washedsuccessively with water (5 ml), 1M aqueous citric acid solution (5 ml)and saturated brine (5 ml), dried (MgSO₄), filtered and evaporated underreduced pressure. The residue was purified by chromatography on silicagel, using an elution gradient of ethyl acetate:hexane (0:100 to 80:20),to furnish the required product (2.4 g, 86%) as a white foam. Rf 0.33(SS 12). Found: C,52.11; H,5.82; N,9.59. C₃₈ H₅₁ F₃ N₆ O₁₀ S; 0.50 CH₂Cl₂ requires C,52.34; H,5.93; N,9.51%. m/e 841.1 (M+H)⁺.

PREPARATION 23 4-Methyl-1-{N-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

A solution of sodium carbonate (1.2 g, 11.3 mmol) in water (15 ml) wasadded dropwise to a stirred solution of the title compound ofPreparation 22 (2.4 g, 2.8 mmol) in methanol (25 ml) at roomtemperature. After 1.5 hours the bulk of the methanol was removed underreduced pressure. The remaining aqueous phase was extracted with ethylacetate (2×10 ml) and the combined organic extracts were dried (MgSO₄),filtered and evaporated under reduced pressure. The residue was purifiedby chromatography on silica gel, using an elution gradient ofdichloromethane:hexane (75:25 to 100:0), followed bydichloromethane:methanol:0.880 aqueous ammonia (95:5:0.5 to 90:10:1), togive the required product (2.07 g, 97%) as a white foam. Rf 0.40 (SS 9).Found: C,55.22; H,6.72; N,10.65. C₃₆ H₅₂ N₆ O₉ S; 0.50 CH₂ Cl₂ requiresC,55.67; H,6.78; N,10.67%.

PREPARATION 24 4-Methyl-1-{N-2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

Aqueous formaldehyde solution (37% w/v, 0.9 ml, 11 mmol) was added to astirred solution of the title compound of Preparation 23 (2.07 g, 2.7mmol) in dichloromethane (20 ml) at room temperature then, after 20minutes, sodium triacetoxyborohydride (0.82 g, 3.8 mmol) was added.After a further 2 hours, the reaction mixture was washed with saturatedaqueous sodium bicarbonate solution (2×10 ml) and saturated brine (10ml), dried (MgSO₄), filtered and evaporated under reduced pressure. Theresidue was purified by chromatography on silica gel, using an elutiongradient of dichloromethane:hexane (75:25 to 100:0), followed bydichloromethane:methanol:0.880 aqueous ammonia (99:1:0.5 to 95:5:0.5),to afford the required product (1.95 g, 93%) as a white foam. Rf 0.54(SS 9). Found: C,55.95; H,7.00; N,10.36. C₃₇ H₅₄ N₆ O₉ S; 0.50 CH₂ Cl₂requires C,56.19; H,6.92; N,10.48%.

PREPARATION 25 4-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester dihydrochloride

A stirred, ice-cooled solution of the title compound of Preparation 24(1.93 g, 2.5 mmol) in dichloromethane (30 ml) was saturated withhydrogen chloride. The solution was allowed to attain room temperatureand after 5 hours the solvent and excess hydrogen chloride were removedunder reduced pressure. Residual traces of hydrogen chloride wereremoved azeotropically with dichloromethane to furnish the product (1.57g, 98%) as a white powder. Rf 0.22 (SS 24). Found: C,47.26; H,6.38;N,11.89. C₂₇ H₃₈ N₆ O₅ S;2HCl; H₂ O; 0.50 CH₂ Cl₂ requires C,47.72;H,6.26; N,12.14%.

PREPARATION 26N-(3-Trifluoroacetyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulfonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alaninet-butyl ester

The title compound (1.9 g, 70%) was obtained from the title compounds ofPreparation 3 (1.62 g, 4.7 mmol) and Preparation 11 (1.36 g, 4.3 mmol),using the procedure described in Preparation 12, as a white foam. Rf0.65 (SS 26). Found C,59.14; H,6.09; N,6.54. C₃₁ H₃₈ F₃ N₃ O₅ S;0.125CH₂ Cl₂ requires C,59.11; H,6.09; N.6.64%. m/e 622.1 (M+H)⁺.

PREPARATION 27N-(3-Trifluoroacetyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulfonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alaninehydrochloride

The title compound (1.85 g, 100%) was obtained from the title compoundof Preparation 26 (1.89 g, 3 mmol), using the procedure described inPreparation 13, as a white foam. Rf 0.35 (SS 13). m/e 566.2 (M+H)⁺.

PREPARATION 28 4-Methyl-1-N-(3-trifluoroacetyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

The title compound (1.7 g, 80%) was obtained from the title compounds ofPreparation 27 (1.82 g, 3.0 mmol) and Preparation 16 (1.1 g, 3.3 mmol),using the procedure of Preparation 20, as a white foam. Rf 0.68 (SS 9).Found: C,58.50; H,5.43; N,7.39. C₃₅ H₄₁ F₃ N₄ O₆ S; 0.10 CH₂ Cl₂requires C,59.10; H,5.83; N,7.87%. m/e 703.3 (M+H)⁺.

PREPARATION 29 4-Methyl-1-N-(3-trifluoroacetyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl)-3-(1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

The title compound (1.24 g, 78%) was obtained from the title compound ofPreparation 28 (1.8 g, 2.5 mmol), using the procedure of Preparation 21,as a tan-coloured foam. Rf 0.21 (SS 9). Found: C,53.43; H,5.35; N,8.72.C₂₈ H₃₅ F₃ N₄ O₆ S; 0.20 CH₂ Cl₂ requires C,53.78; H,5.66; N,8.89%. m/e613.3 (M+H)⁺.

PREPARATION 30 4-Methyl-1-{N-3-trifluoroacetyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

The title compound (1.49 g, 87%) was obtained from the title compound ofPreparation 29 (1.22 g, 2 mmol), using the procedure of Preparation 22,as a white foam. Rf 0.84 (SS 26). Found: C,52.72; H,5.81; N,9.36. C₃₉H₅₃ F₃ N₆ O₁₀ S; 0.50 CH₂ Cl₂ requires C,52.86; H,6.06; N,9.36%.

PREPARATION 31 4-Methyl-1-{N -2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

The title compound (0.94 g, 72%) was obtained from the title compound ofPreparation 30 (1.47 g, 1.7 mmol), using the procedure of Preparation23, as a white foam. Rf 0.38 (SS 9). Found: C,56.08; H,7.22; N,10.15.C₃₇ H₅₄ N₆ O₉ S; 0.50 CH₂ Cl₂ requires C,56.19; H,6.91; N,10.48%.

PREPARATION 32 4-Methyl-1-{N-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester

The title compound (0.92 g, 97%) was obtained from the title compound ofPreparation 31 (0.93 g, 1.2 mmol), using the procedure of Preparation24, as a white foam. Rf 0.49 (SS 9). Found: C,56.45; H,7.18; N,9.89. C₃₈H₅₆ N₆ O₉ S; H₂ O; 0.33 CH₂ Cl₂ requires C,56.19; H,7.21; N,10.25%. m/e773.4 (M+H)⁺.

PREPARATION 33 4-Methyl-1-N-(3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid methyl ester dihydrochloride

The title compound (0.71 g, 83%) was obtained from the title compound ofPreparation 32 (0.92 g, 1.2 mmol), using the procedure of Preparation25, as a white powder. Rf 0.39 (SS 28). Found: C,48.11; H,6.88; N,11.44.C₂₈ H₄₀ N₆ O₅ S; 2HCl; 0.50 CH₂ Cl₂ requires C,47.87; H,6.48; N,11.75%.m/e 573.4 (M+H)⁺.

PREPARATION 34 1-N-t-Butoxycarbonyl-3-(3-pyridyl)-(S)-alanyl!-4(R)-methylpiperidine-2(R)-carboxylicacid ethyl ester

A stirred, ice-cooled suspension ofN-t-butoxycarbonyl-3-(3-pyridyl)-(S)-alanine (Preparation 7; 2 g, 7.5mmol) in dichloromethane (20 ml) was treated sequentially with1-hydroxybenzotriazole (1.4 g, 9.1 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2 g, 10.4mmol). After 10 minutes, the hydrochloride salt of the title compound ofPreparation 14 (1.7 g, 8.2 mmol) was added as a solution indichloromethane (5 ml), dropwise, followed by the dropwise addition of asolution of N-methylmorpholine (0.9 g, 9 mmol) in dichloromethane (3ml). After 72 hours at room temperature, the solvent was evaporatedunder reduced pressure. The residue was dissolved in ethyl acetate (50ml) and this solution was washed successively with saturated aqueoussodium bicarbonate solution and saturated brine, dried (MgSO₄), filteredand evaporated under reduced pressure. The crude product was purified bychromatography on silica gel, using an elution gradient of ethylacetate:hexane (10:90 to 100:0), to afford the title compound (2.1 g,67%) as a hygroscopic foam. Rf 0.40 (SS 26). m/e 420.2 (M+H)⁺.

PREPARATION 35 1-N-t-Butoxycarbonyl-3-(1-benzyl-3-pyridinium)-(S)-alanyl!-4(R)-methylpiperidine-2(R)-carboxylicacid ethyl ester bromide

To a solution of the title compound of Preparation 34 (3.3 g, 7.8 mmol)in dry ethanol (20 ml) was added a solution of benzyl bromide (2.7 g,15.7 mmol) in dry ethanol (10 ml), dropwise, over 5 minutes at roomtemperature. After 18 hours the ethanol was evaporated under reducedpressure. The residue was dissolved in acetonitrile (30 ml), then thesolution extracted with hexane (3×10 ml) and evaporated to dryness underreduced pressure. Solvent traces were removed azeotropically from theresidue with dichloromethane to give the title compound (4.64 g, 100%)which was used without further purification.

PREPARATION 36 1-N-t-Butoxycarbonyl-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-4(R)-methylpiperidine-2(R)-carboxylicacid ethyl ester

Sodium borohydride (0.69 g, 18 mmol) was added in two equal portionsover 5 minutes to a stirred, ice-cooled solution of the title compoundof Preparation 35 (3.58 g, 6 mmol) in ethanol (40 ml). After 1 hour thesolution was evaporated to dryness under reduced pressure. The residuewas dissolved in dichloromethane (50 ml) and the solution washed withsaturated aqueous sodium bicarbonate solution (2×15 ml) and saturatedbrine (15 ml), dried (MgSO₄), filtered and evaporated under reducedpressure. The crude product was purified by chromatography on silicagel, using an elution gradient of dichloromethane:hexane (75:25 to100:0), followed by dichloromethane:methanol:0.880 aqueous ammonia(99:1:0.5 to 95:5:0.5), to furnish the title compound (3.18 g, 80%) as agum. Rf 0.80 (SS 9). m/e 514.4 (M+H)⁺.

PREPARATION 37 1-3-(1-Benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-4(R)-methylpiperidine-2(R)-carboxylicacid ethyl ester dihydrochloride

A stirred, ice-cooled solution of the title compound of Preparation 36(3.1 g, 6 mmol) in dichloromethane (40 ml) was saturated with hydrogenchloride. After 1.5 hours the solvent and excess hydrogen chloride wereremoved under reduced pressure. Residual traces of hydrogen chloridewere removed azeotropically from the residue with dichloromethane toafford the title compound (3 g, 100%) as a hygroscopic foam, which wasused without further purification. Rf 0.42 (SS 9). m/e 414.3 (M+H)⁺.

PREPARATION 38 4(R)-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid ethyl ester

A solution of N-ethyldiisopropylamine (1.6 g, 12.4 mmol) indichloromethane (5 ml) was added dropwise over 5 minutes to a stirredice-cooled suspension of the title compound of Preparation 37 (1.5 g, 3mmol) and 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulfonylchloride (Preparation 1; 1.1 g, 3.3 mmol) in dichloromethane (15 ml).After 18 hours at room temperature the solvent was evaporated underreduced pressure and the residue dissolved in ethyl acetate (50 ml).This solution was washed with water (2×10 ml), saturated aqueous sodiumbicarbonate solution (2×10 ml) and saturated brine (10 ml), dried(MgSO₄), filtered and evaporated under reduced pressure. The residue waspurified by chromatography on silica gel, using an elution gradient ofethyl acetate:hexane:diethylamine (0:100:1 to 40:60:1), to yield thetitle compound (1 g 48%) as a pale yellow foam. Rf 0.70 (SS 9). Found:C,58.95; H,5.83; N,7.47 C₃₅ H₄₃ F₃ N₄ O₆ S requires C,59.64; H,6.15;N,7.95%. m/e 705.4 (M+H)⁺.

PREPARATION 39 4(R)-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid ethyl ester

The title compound (0.76 g, 84%) was obtained from the title compound ofPreparation 38 (1 g,1.4 mmol), using the procedure of Preparation 21, asa white foam. Rf 0.25 (SS 9). Found: C,53.88; H,5.77; N,8.77. C₂₈ H₃₇ F₃N₄ O₆ S; 0.20 CH₂ Cl₂ requires C,53.61; H,5.97; N,8.87%. m/e 615.4(M+H)⁺.

PREPARATION 40 4(R)-Methyl-1-{N-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(R)-carboxylicacid ethyl ester

The title compound (0.48 g, 47%) was obtained from the title compound ofPreparation 39 (0.74 g, 1.2 mmol), using the procedure of Preparation22, as a white foam and used without further purification. Rf 0.42 (SS12). m/e 857.5 (M+H)⁺.

PREPARATION 41 4(R)-Methyl-1-{N-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(R)-carboxylicacid ethyl ester

The title compound (0.43 g, 100%) was obtained from the title compoundof Preparation 40 (0.47 g, 0.5 mmol), using the procedure of Preparation23, as a white foam. Rf 0.44 (SS 9). m/e 761.5 (M+H)⁺.

PREPARATION 42 4(R)-Methyl-1-{N-2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(R)-carboxylicacid ethyl ester

The title compound (0.34 g, 82%) was obtained from the title compound ofPreparation 41 (0.41 g, 0.5 mmol), using the procedure of Preparation24, as a white foam. Rf 0.57 (SS 9). Found: C,58.42; H,7.67; N,9.98. C₃₈H₅₈ N₆ O₉ S requires C,58.89; H,7.54; N,10.84%. m/e 775.4 (M+H)⁺.

PREPARATION 43 4(R)-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid ethyl ester hydrochloride

The title compound (0.18 g, 66%) was obtained from the title compound ofPreparation 42 (0.33 g, 0.4 mmol), using the procedure of Preparation25, as a white powder. Rf 0.48 (SS 27). Found: C, 52.49; H,7.18;N,12.94. C₂₈ H₄₂ N₆ O₅ S; HCl; H₂ O; 0.20 CH₂ Cl₂ requires: C,52.41;H,7.08; N, 13.00%. m/e 575.4 (M+H)⁺.

PREPARATION 44 4-Methyl-1,2,3,6-tetrahydropyridine-2(S)-carboxylic acidmethyl ester p-toluenesulphonate

The title compound is obtainable via 4-methyl-1-1-(R)-phenylethyl!-1,2,3,6-tetrahydropyridine-2(S)-carboxylic acidmethyl ester by analogy with Preparation 16.

Alternatively, it may be obtained via 4-methyl-1-1-(S)-phenylethyl!-1,2,3,6-tetrahydropyridine-2(S)-carboxylic acidmethyl ester hydrochloride which is a by-product of Preparation 16, bysubsequent processing as described in Preparation 16.

PREPARATION 45 4(R)-Methyl-2(S)-carboxylic acid methyl esterp-toluenesulphonate

A stirred solution of the title compound of Preparation 44 (2.0 g, 6.1mmol) in methanol (20 ml) was hydrogenated over 10% palladium oncharcoal at 414 kPa (60 p.s.i.) and room temperature for 20 hours, thenthe mixture filtered. The filtrate was evaporated under reduced pressureand the residue crystallised from 2-propanol to provide the titlecompound (1.2 g, 60%) as white crystals. Rf 0.80 (SS 28). Found:C,54.51; H,7.28; N,3.94. C₈ H₁₅ NO₂ ; C₇ H₈ O₃ S requires C,54.69;H,7.04; N,4.25%.

PREPARATION 46 4(S)-Methyl-2(R)-carboxylic acid methyl esterp-toluenesulphonate

The title compound (2.0 g, 77%) was obtained from the title compound ofPreparation 16, using the procedure of Preparation 45, as whitecrystals. Rf 0.80 (SS 28). Found: C,54.44; H,7.02; N,4.60. C₈ H₁₅ NO₂ ;C₇ H₈ O₃ S requires C,54.69; H,7.04; N,4.25%.

PREPARATION 47 4-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(S)-carboxylic acid methyl ester

The title compound (1.75 g, 82%) was obtained from the title compoundsof Preparation 13 (1.82 g, 3.1 mmol) and Preparation 44 (1.1 g, 3.4mmol), using the procedure of Preparation 20, as a white foam. Rf 0.41(SS 9). Found: C,57.42; H,5.20; N,7.87. C₃₄ H₃₉ F₃ N₄ O₆ S; 0.33 CH₂ Cl₂requires C,57.50; H,5.57; N,7.81 %. m/e 689.2 (M+H)⁺.

PREPARATION 48 4(R)-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(S)-carboxylicacid methyl ester

The title compound (2.09 g, 95%) was obtained from the title compoundsof Preparation 13 (1.88 g, 3.19 mmol) and Preparation 45 (1.15 g, 3.49mmol), using the procedure of Preparation 20, as a white foam. Rf 0.52(SS 26). Found: C,58.58; H,5.81; N,7.77. C₃₄ H₄₁ F₃ N₄ O₆ S; 0.075 CH₂Cl₂ requires C,58.71; H,5.95; N,8.04%. m/e 691.3 (M+H)⁺.

PREPARATION 49 4(S)-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-benzyl-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid methyl ester

The title compound (2.3 g, 92%) was obtained from the title compounds ofPreparation 13 (2.13 g, 3.6 mmol) and Preparation 46 (1.3 g, 3.9 mmol),using the procedure of Preparation 20, as a white foam. Rf 0.50 (SS 26).Found: C,58.80; H,5.89; N,7.98. C₃₄ H₄₁ F₃ N₄ O₆ S requires C,59.12;H,5.98; N,8.11%. m/e 691.4 (M+H)⁺.

PREPARATION 50 4-Methyl-1-N-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid methyl ester

The title compound (1.24 g, 83%) was obtained from the title compound ofPreparation 47 (1.72 g, 2.5 mmol), using the procedure of Preparation21, as a white foam. Rf 0.24 (SS 9). m/e 599.2 (M+H)⁺.

PREPARATION 51 4(R)-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(S)-carboxylicacid methyl ester hydrochloride

The title compound (1.47 g, 84%) was obtained from the title compound ofPreparation 48 (2.0 g, 2.89 mmol), using the procedure of Preparation21, as a pale yellow foam. Rf 0.30 (SS 9). Found: C,51.36; H,5.32;N,8.81. C₂₇ H₃₅ F₃ N₄ O₆ S; HCl requires C,50.90; H,5.70; N,8.79%. m/e601.3 (M+H)⁺.

PREPARATION 52 4(S)-Methyl-1-N-(2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid methyl ester hydrochloride

The title compound (1.79 g, 89%) was obtained from the title compound ofPreparation 49 (2.3 g, 3.3 mmol), using the procedure of Preparation 21,as a pale yellow foam. Rf 0.45 (SS 9). m/e 601.3 (M+H)⁺.

PREPARATION 53 4-Methyl-1-{N-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid methyl ester

The title compound (1.47 g, 84%) was obtained from the title compound ofPreparation 50 (1.24 g, 2.1 mmol), using the procedure of Preparation22, as a white foam. Rf 0.25 (SS 12). Found: C,53.04; H,5.79; N,9.35.C₃₈ H₅₁ F₃ N₆ O₁₀ S; 0.33 CH₂ Cl₂ requires C,52.96; H,5.99; N,9.66%. m/e841.3 (M+H)⁺.

PREPARATION 54 4(R)-Methyl-1-{N-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(S)-carboxylicacid methyl ester

The title compound (1.52 g, 75%) was obtained from the title compound ofPreparation 51 (1.45 g, 2.41 mmol), using the procedure of Preparation22, as a white foam. Rf 0.60 (SS 29)

PREPARATION 55 4(S)-Methyl-1-{N-2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(R)-carboxylicacid methyl ester

The title compound (2.17 g, 88%) was obtained from the title compound ofPreparation 52 (1.76 g, 2.9 mmol), using the procedure of Preparation22, as a white foam. Rf 0.72 (SS 9).

PREPARATION 56 4-Methyl-1-{N-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid methyl ester

The title compound (1.11 g, 86%) was obtained from the title compound ofPreparation 53 (1.46 g, 1.74 mmol), using the procedure of Preparation23, as a white foam. Rf 0.43 (SS 9). m/e 745.3 (M+H)⁺.

PREPARATION 57 4(R)-Methyl-1-{N-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(S)-carboxylicacid methyl ester

The title compound (850 mg, 64%) was obtained from the title compound ofPreparation 54 (1.5 g, 1.78 mmol), using the procedure of Preparation23, as a white foam. Rf 0.60 (SS 9). Found: C, 56.18; H,7.09; N,10.64.C₃₆ H₅₄ N₆ O₉ S; 0.30 CH₂ Cl₂ requires C,56.45; H,7.13; N,10.88%.

PREPARATION 58 4(S)-Methyl-1-{N-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(R)-carboxylicacid methyl ester

The title compound (1.17 g, 61%) was obtained from the title compound ofPreparation 55 (2.17 g, 2.6 mmol), using the procedure of Preparation23, as a white foam. Rf 0.45 (SS 9). m/e 747.5.

PREPARATION 59 4-Methyl-1-{N-2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid methyl ester

The title compound (980 mg, 86%) was obtained from the title compound ofPreparation 56 (1.11 g, 1.4 mmol), using the procedure of Preparation24, as a white foam. Rf 0.59 (SS 25). Found: C,56.21; H,7.09; N,10.19.C₃₇ H₅₄ N₆ O₉ S; 0.50 CH₂ Cl₂ requires C,56.19; H,6.92; N,10.48%.

PREPARATION 60 4(R)-Methyl-1-{N-2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(S)-carboxylicacid methyl ester

The title compound (700 mg, 83%) was obtained from the title compound ofPreparation 57 (830 mg, 1.11 mmol), using the procedure of Preparation24, as a white foam. Rf 0.61 (SS 9). Found: C,58.38; H,7.50; N,10.51.C₃₇ H₅₆ N₆ O₉ S requires C,57.91; H,7.36; N,10.92. m/e 762 (M+H)⁺.

PREPARATION 61 4(S)-Methyl-1-{N-2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl!-3-1-(N,N'-di-t-butoxycarbonylamidino)-1,2,5,6-tetrahydro-3-pyridyl!-(S)-alanyl}piperidine-2(R)-carboxylicacid methyl ester

The title compound (1.0 g, 98%) was obtained from the title compound ofPreparation 58 (1.0 g, 1.3 mmol), using the procedure of Preparation 24,as a white foam. Rf 0.48 (SS 9). Found: C,58.23; H,7.55; N,10.64. C₃₇H₅₆ N₆ O₉ S requires C,57.91; H,7.36; N,10.92%.

PREPARATION 62 4-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(S)-carboxylicacid methyl ester hydrochloride

The title compound (576 mg, 76%) was obtained from the title compound ofPreparation 59 (970 mg, 1.28 mmol), using the procedure of Preparation25, as a foam. Rf 0.41 (SS 27). Found: C,50.37; H,6.70; N,12.91. C₂₇ H₃₈N₆ O₅ S; HCl; 2H₂ O; 0.25 CH₂ Cl₂ requires C,50.16; H,6.72; N,12.88%.m/e 559.4 (M+H)⁺.

PREPARATION 63 4(R)-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(S)-carboxylic acid methyl ester hydrochloride

The title compound (360 mg, 62%) was obtained from the title compound ofPreparation 60 (700 mg, 0.92 mmol), using the procedure of Preparation25, as a yellow foam. Rf 0.56 (SS 27). m/e 561.4 (M+H)⁺.

PREPARATION 64 4(S)-Methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid methyl ester dihydrochloride

The title compound (810 mg, 98%) was obtained from the title compound ofPreparation 61 (1.0 g, 1.3 mmol), using the procedure of Preparation 25,as a white foam. Rf 0.48 (SS 27). Found: C,51.40; H,6.97; N,13.55. C₂₇H₄₀ N₆ O₅ S; 2HCl requires C,51.18; H,6.68; N,13.20%.

Biological activity

The following Table illustrates the in vitro inhibitory activitiesagainst thrombin and trypsin for two of the compounds of the invention.

                  TABLE                                                           ______________________________________                                                     ki(M)                                                            EXAMPLE        THROMBIN  TRYPSIN                                              ______________________________________                                        5              1.9 × 10.sup.-9                                                                   2.4 × 10.sup.-6                                6              9.9 × 10.sup.-9                                                                   6.9 × 10.sup.-7                                ______________________________________                                    

Safety profile

Certain compounds of the invention have been tested at multiple doses ofup to 30 mg/kg i.v. in mouse and up to 100 mg/kg i.v. in rat withoutshowing any sign of adverse toxicity.

I claim:
 1. A compound of formula (I): ##STR26## or a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable solvate ofeither entity,wherein Y is optionally monounsaturated C₃ -C₅ alkyleneoptionally substituted with C₁ -C₄ alkyl or methylene; R¹ is H; C₁ -C₄alkyl optionally substituted with C₁ -C₄ alkoxy, OH, NR⁵ R⁶, CONR⁵ R⁶,C₃ -C₆ cycloalkyl or aryl; or C₃ -C₆ alkenyl; R² is H; C₁ -C₄ alkyloptionally substituted with C₁ -C₄ alkoxy, OH, NR⁵ R⁶, CONR⁵ R⁶, C₃ -C₆cycloalkyl or aryl; or CONR⁵ R⁶ ; R³ and R⁴ are each independentlyselected from H; C₁ -C₄ alkyl optionally substituted with NR⁵ R⁶ ; C₁-C₄ alkoxy; halo; CONR⁵ R⁶ and aryl; aryl is phenyl optionallysubstituted with one, two or three substituents independently selectedfrom C₁ -C₄ alkyl, C₁ -C₄ alkoxy, OH, halo and CF₃ ; R⁵ and R⁶ are eachindependently selected from H and C₁ -C₄ alkyl; and m and n are eachindependently 1, 2 or
 3. 2. A compound according to claim 1 wherein thepreferred stereoisomer is of formula (IA): ##STR27##
 3. A compoundaccording to claim 2 wherein Y is optionally monounsaturated C₄ alkylenesubstituted with methyl or ethyl; R¹ is C₁ -C₄ alkyl; R² is H; R³ and R⁴are H; and m and n are each independently 1 or
 2. 4. A compoundaccording to claim 3 of formula (IB): ##STR28## wherein -- represents anoptional carbon-carbon single bond; R¹ and R⁷ are methyl; m is 1 or 2;and n is
 2. 5. A compound according to claim 4 wherein the compound offormula (IB) is selected from4-methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid; 4-methyl-1-N-(3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid; and 4(R)-methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid;and pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable solvates of either entity.
 6. A pharmaceutical compositioncomprising a compound of claim 1, a pharmaceutically acceptable saltthereof or a pharmaceutically acceptable solvate of said compound orsaid salt, and a pharmaceutically acceptable carrier or diluent.
 7. Acompound of formula (II): t,0770wherein Y is optionally monounsaturatedC₃ -C₅ alkylene optionally substituted with C₁ -C₄ alkyl or methylene;R¹ is H; C₁ -C₄ alkyl optionally substituted with C₁ -C₄ alkoxy, OH, NR⁵R⁶, CONR⁵ R⁶, C₃ -C₆ cycloalkyl or aryl; or C₃ -C₆ alkenyl; R² is H; C₁-C₄ alkyl optionally substituted with C₁ -C₄ alkoxy, OH, NR⁵ R₆,CONR5R⁶, C₃ -C₆ cycloalkyl or aryl; or CONR⁵ R⁶ ; R³ and R⁴ are eachindependently selected from H; C₁ -C₄ alkyl optionally substituted withNR⁵ R⁶ ; C₁ -C₄ alkoxy; halo; CONR⁵ R⁶ and aryl; aryl is phenyloptionally substituted with one, two or three substituents independentlyselected from C₁ -C₄ alkyl, C₁ -C₄ alkoxy, OH, halo and CF₃ ; R⁵ and R⁶are each independently selected form H and C₁ -C₄ alkyl; m and n areeach independently 1, 2 or 3; and R⁸ is C₁ -C₃ alkyl.
 8. A compoundaccording to claim 7 wherein R⁸ is methyl or ethyl.
 9. A method oftreating a mammal to cure or prevent deep vein thrombosis (DVT) aftersurgery, major medical illness, paralysis, malignancy, prolongedimmobilization trauma, application of lower limb plaster casts, orfractures of the lower limbs or pelvis; recurrent DVT; DVT duringpregnancy when there is a previous history thereof; reocclusionfollowing thrombolytic therapy; chronic arterial obstruction; peripheralvascular disease; acute myocardial infarction; unstable angina; atrialfibrillation; thrombotic stroke; transient ischaemic attacks;disseminated intravascular coagulation; coagulation in extra-corporealcircuits; occlusion of arterio-venous shunts and blood vessel grafts(including coronary artery by-pass grafts); restenosis and occlusionfollowing angioplasty; neurodegenerative disorders; inflammatorydisorders; or scarring; which comprises administering to said mammal aneffective amount of a compound of claim 1, a pharmaceutically acceptablesalt thereof, or a pharmaceutically acceptable solvate of said compoundor said salt or a pharmaceutical composition containing any of theforegoing.
 10. A process for the preparation of a compound of formula(I): ##STR29## or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable solvate of either entity,wherein Y isoptionally monounsaturated C₃ -C₅ alkylene optionally substituted withC₁ -C₄ alkyl or methylene; R¹ is H; C₁ -C₄ alkyl optionally substitutedwith C₁ -C₄ alkoxy, OH, NR⁵ R⁶, CONR⁵ R⁶, C₃ -C₆ cycloalkyl or aryl; orC₃ -C₆ alkenyl; R² is H; C₁ -C₄ alkyl optionally substituted with C₁ -C₄alkoxy, OH, NR⁵ R⁶, CONR⁵ R⁶, C₃ -C₆ cycloalkyl or aryl; or CONR⁵ R⁶ ;R³ and R⁴ are each independently selected from H; C₁ -C₄ alkyloptionally substituted with NR⁵ R⁶ ; C₁ -C₄ alkoxy; halo; CONR⁵ R⁶ andaryl; aryl is phenyl optionally substituted with one, two or threesubstituents independently selected from C₁ -C₄ alkyl, C₁ -C₄ alkoxy,OH, halo and CF₃ ; R⁵ and R⁶ are each independently selected from H andC₁ -C₄ alkyl; and m and n are each independently 1, 2 or 3;whichcomprises acid- or base-catalysed hydrolysis of a compound of formula(II): ##STR30## wherein R⁸ is C₁ -C₃ alkyl, and Y, R¹, R², R³, R⁴, m andn are as previously defined in this claim; followed by optionalformation of a pharmaceutically acceptable salt of the required productor a pharmaceutically acceptable solvate of either entity.
 11. A processaccording to claim 10 which is base-catalysed in aqueous medium,optionally in the presence of a cosolvent, at from about 0° C. to about100° C.
 12. A process according to claim 11 wherein the base is analkali metal hydroxide.
 13. A process according to claim 12 wherein thebase is sodium hydroxide and the reaction is conducted at from about 0°C. to about room temperature.
 14. A process according to claim 13wherein R⁸ is methyl or ethyl.
 15. A process according to claim 14wherein the preferred stereoisomer of a compound of formula (I) producedform the corresponding stereoisomer of a compound of formula (II) is offormula (IA): ##STR31##
 16. A process according to claim 15 where Y isoptionally monounsaturated C₄ alkylene substituted with methyl or ethyl;R¹ is C₁ -C₄ alkyl; R² is H; R³ and R⁴ are H; and m and n are eachindependently 1 or
 2. 17. A process according to claim 16 wherein thecompound produced is of formula (IB): ##STR32## wherein -- represents anoptional carbon-carbon single bond; R¹ and R⁷ are methyl; m is 1 or 2;and n is
 2. 18. A process according to claim 17 wherein the compound offormula (IB) produced is selected from4-methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid; 4-methyl-1-N-(3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!-1,2,3,6-tetrahydropyridine-2(R)-carboxylicacid; and 4(R)-methyl-1-N-(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-sulphonyl)-3-(1-amidino-1,2,5,6-tetrahydro-3-pyridyl)-(S)-alanyl!piperidine-2(R)-carboxylicacid;and pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable solvates of either entity.